Note: Descriptions are shown in the official language in which they were submitted.
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Novel cellular glycan compositions
FIELD OF THE INVENTION
The invention describes novel compositions of glycans, glycomes, from human
multipotent stem
cells, and especially novel subcompositions of the glycomes with specific
monosaccharide
compositions and glycan structures. The invention is further directed to
methods for modifying the
glycomes and analysis of the glycomes and the modified glycomes. Furthermore,
the invention is
directed to stem cells carrying the modified glycomes on their surfaces. The
glycomes are
preferably analysed by profiling methods able to detect reproducibly and
quantitatively numerous
individual glycan structures at the same time. The most preferred type of the
profile is a mass
spectrometric profile. The invention specifically revealed novel target
structures and is especially
directed to the development of reagents recognizing the structures.
BACKGROUND OF THE INVENTION
Stem Cells
Stem cells are undifferentiated cells which can give rise to a succession of
mature functional cells.
For example, a hematopoietic stem cell may give rise to any of the different
types of terminally
differentiated blood cells. Embryonic stem (ES) cells are derived from the
embryo and are
pluripotent, thus possessing the capability of developing into any organ or
tissue type or, at least
potentially, into a complete embryo.
The first evidence for the existence of stem cells came from studies of
embryonic carcinoma (EC)
cells, the undifferentiated stem cells of teratocarcinomas, which are tumors
derived from germ cells.
These cells were found to be pluripotent and immortal, but possess limited
developmental potential
and abnormal karyotypes (Rossant and Papaioannou, Cell Differ 15,155-161,
1984). The glycans of
cancer cells change by frequent mutations and the data from the cancer cell
lines is not valid for ES
cells. ES cells, on the other hand, are thought to retain greater
developmental potential because they
are derived from normal embryonic cells, without the selective pressures of
the teratocarcinoma
environment.
Pluripotent embryonic stem cells have traditionally been derived principally
from two embryonic
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sources. One type can be isolated in culture from cells of the inner cell mass
of a pre-implantation
embryo and are termed embryonic stem (ES) cells (Evans and Kaufman, Nature
292,154-156, 1981;
U.S. Pat. No. 6,200,806). A second type of pluripotent stem cell can be
isolated from primordial
germ cells (PGCS) in the mesenteric or genital ridges of embryos and has been
termed embryonic
germ cell (EG) (U.S. Pat. No. 5,453,357, U.S. Pat. No. 6,245,566). Both human
ES and EG cells are
pluripotent. This has been shown by differentiating cells in vitro and by
injecting human cells into
immunocompromised (SCUM) mice and analyzing resulting teratomas (U.S. Pat. No.
6,200,806).
The term "stem cell" as used herein means stem cells including embryonic stem
cells or embryonic
type stem cells and stem cells diffentiated thereof to more tissue specific
stem cells.
The present invention provides novel markers and target structures and binders
to these for
especially embryonic stem cells. From hematopoietic CD34+ cells certain
terminal structures such
as terminal sialylated type two N-acetyllactosamines such as
NeuNAc0Gal04G1cNAc (Magnani J.
US6362010 ) has been suggested and there is indications for low expression of
Slex type structures
NeuNAca3Ga104(Fuca3)G1cNAc (Xia L et al Blood (2004) 104 (10) 3091-6). The
invention is
also directed to the NeuNAca3Ga1(34G1cNAc non-polylactosamine variants
separately from
specific characteristic 0-glycans and N-glycans. Due to tissue specificity of
glycosylation such data
is not relevant to embryonic stem cells, which represent much earlier level of
differentiation.
Human ES, EG and EC cells, as well as primate ES cells, express alkaline
phosphatase, the stage-
specific embryonic antigens SSEA-3 and SSEA-4, and surface proteoglycans that
are recognized by
the TRA-1-60; and TRA-1-81 antibodies. All these markers typically stain these
cells, but are not
entirely specific to stem cells, and thus cannot be used to isolate stem cells
from organs or
peripheral blood.
The SSEA-3 and SSEA-4 structures are known as galactosylgloboside and
sialylgalactosylgloboside, which are among the few suggested structures on
embryonic stem cells,
though the nature of the structures in not ambigious. An antibody called K21
has been suggested to
bind a sulfated polysaccharide on embryonic carcinoma cells (Badcock G et
alCancer Res (1999)
4715-19. Due to cell type, species, tissue and other specificity aspects of
glycosylation (Furukawa,
K., and Kobata, A. (1992) Curr. Opin. Struct. Biol. 3, 554-559, Gagneux, and
Varki, A. (1999)
Glycobiology 9, 747-755;Gawlitzek, M. et al. (1995), J. Biotechnol. 42, 117-
131; Goelz, S.,
Kumar, R., Potvin, B., Sundaram, S., Brickelmaier, M., and Stanley, P. (1994)
J. Biol. Chem. 269,
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1033-1040; Kobata, A (1992) Eur. J. Biochem. 209 (2) 483-501.) This result
does not indicate the
presence of the structure on native embryonic stem cells. The present
invention is directed to human
stem cells.
Some low specificity plant lectin reagents have been reported in binding of
embryonic stem cell like
materials. Venable et al 2005, (Dev. Biol. 5:15) measured lectins the binding
of SSEA-4 antibod
positive subpopulation of embryonic stem cells. This approach suffers obvious
problems. It does
not tell the expression of the structures in antive non-selected embryonic
strem cells. The SSEA-4
was chosen select especially pluripotent stem cells. The scientists of the
same Bresagen company
have further revealed that actual role of SSEA-4 with the specific stem cell
lines is not relevant for
the pluripotency.
The work does not reveal: 1) The actual amount of molecules binding to the
lectins or 2) presence
of any molecules due to defects caused by the cell sorting and experimental
problems such as
trypsination of the cells. It is really alerting that the cells were
trypsinized, which removes protein
and then enriched by possible glycolipid binding SSEA4 antibody and secondary
antimouse
antibody, fixed with paraformaldehyde without removing the antibodies, and
labelled by
simultaneous with lectin and the same antibody and then the observed glycan
profile is the similar
as revealed by lectin analysis by same scientist for antibody glycosylation
(M. Pierce US2005 ) or
3) the actual structures, which are bound by the lectins. To reveal the
possible residual binding to
the cells would require analysis of of the glycosylations of the antibodies
used (sources and lots not
revealed).
The purity of the SSEA-4 positive cells was reported to be 98-99 %, which is
unusually high. The
quantitation of the binding is not clear as figure 18 shows about 10 % binding
by lectins LTL and
DBA, which are not bound to hESC-cells 3rd page, column 2, paragraph 2 and by
immunocytochemistry 4the page last line.
It appears that skilled artisan would consider the results of Venable et al
such convienent
colocalization of SSEA-4 and the lectin binding by binding of the lectins to
the anti-SSEA-4
antibody. It appears that the more rare binding would reflect lower proportion
of the terminal
epitope per antibody molecule leading to lower density of the labellable
antibodies. It is also
realized that the non-controlled cell culture process with animal derived
material would lead to
contamination of the cells by N-glycolyl-neuraminic acid, which may be
recognized by anti-mouse
antibodies used as secondary antibody (not defined what kind of anti-mouse)
used in purification
and analysis of purity, which could lead to convieniently high cell purity.
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The work is directed only to the "pluripotent" embryonic stem cells associated
with SSEA-4
labelling and not to differentiated variants thereof as the present invention.
The results indicated
possible binding (likely on the antibodies) to certain potential
monosaccharide epitopes (6Ih page,
Table 21,, and column 2 ) such Gal and Galactosamine for RCA (ricin, inhitable
by Gal or
lactose), G1cNAc for TL (tomato lectin), Man or Glc for ConA, Sialic
acid/Sialic acid a6Ga1NAc
for SNA, Mana for HHL; lectins with partial binding not correlating with SSEA-
4:
Ga1NAc/Ga1NAc(34Ga1(in text) WFA, Gal for PNA, and Sialic acid/Sialic acid
a6Ga1NAc for
SNA; and lectins associated by part of SSEA-4 cells were indicated to bind Gal
by PHA-L and
PHA-E, Ga1NAc by VVA and Fuc by UEA, and Gal by MAA (inhibited by lactose).
UEA binding
was discussed with reference as endothelial marker and 0-linked fucose which
is directly bound to
Ser (Thr) on protein. The background has indicated a H type 2 specificity for
the endothelial UEA
receptor. The specifities of the lectins are somawhat unusual, but the product
codes or isolectin
numbers/names of the lectins were not indicated (except for PHA-E and PHA-L)
and it is known
that plants contain numerous isolectins with varying specificities.
Weame KA et al Glycobiology (2006) 16 (10) 981-990 studied also staining of
embryonic stem
cells by plant lectins. The data using the low specificity reagents does not
reveal exact glycan
structures and specifically not the elongated structure on specific glycan
core structures as described
by the present invention for human embryonic stem cells nor useful antibody
reagent specificities
for specific recognition of terminal epitopes. The authors guess some
binding/non-binding
structures based on the lectin bindings, which appear to be at least partially
different from ones
revealed by the invention indicating possible technical problems. This work
does not imply any
other type of usefulness of the lectins in other cell/cell materials directed
methods. The Wearne data
describes embryonic bodies, which is stage 2 differentiation in present work,
but appears to lack
data about further differentiated cells such as stage 3 cells.
The present invention revealed specifc structures by mass spectrometric
profiling, NMR
spectrometry and binding reagents including glycan modifying enzymes. The
lectins are in general
low specificity molecules. The present invention revealed binding epitiopes
larger than the
previously described monosaccharide epitopes. The larger epitopes allowed us
to design more
specific binding substances with typical binding specificities of at least
disaccharides. The invention
also revealed lectin reagents with speficified with useful specificities for
analysis of native
embryonic stem cells without selection against an uncontrolled marker and/or
coating with an
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antibody or two from different species. Clearly the binding to native
embryonic stem cells is
different as the binding with MAA was clear to most of cells, there was
differences between cell
line so that RCA, LTA and UEA was clearly binding a HESC cell line but not
another.
Methods for separation and use of stem cells are known in the art.
There have been great efforts toward isolating pluripotent or multipotent stem
cells, in earlier
differentiation stages than hematopoietic stem cells, in substantially pure or
pure form for diagnosis,
replacement treatment and gene therapy purposes. Stem cells are important
targets for gene therapy,
where the inserted genes are intended to promote the health of the individual
into whom the stem
cells are transplanted. In addition, the ability to isolate stem cells may
serve in the treatment of
lymphomas and leukemias, as well as other neoplastic conditions where the stem
cells are purified
from tumor cells in the bone marrow or peripheral blood, and reinfused into a
patient after
myelosuppressive or myeloablative chemotherapy.
Multiple adult stem cell populations have been discovered from various adult
tissues. In addition to
hematopoietic stem cells, neural stem cells were identified in adult mammalian
central nervous
system (Ourednik et al. Clin. Genet. 56, 267, 1999). Adult stem cells have
also been identified from
epithelial and adipose tissues (Zuk et al. Tissue Engineering 7, 211, 2001).
Recent studies have
demonstrated that certain somatic stem cells appear to have the ability to
differentiate into cells of a
completely different lineage (Pfendler KC and Kawase E, Obstet Gynecol Surv
58, 197-208, 2003).
Monocyte derived (Zhao et al. Proc. Natl. Acad. Sci. USA 100, 2426-2431, 2003)
and mesodermal
derived (Schwartz et al. J. Clin. Invest 109, 1291-1301, 2002) cells that
possess some multipotent
characteristics were identified. The presence of multipotent "embryonic-like"
progenitor cells in
blood was suggested also by in-vivo experiments following bone marrow
transplantations (Zhao et
al. Brain Res Protoc 11, 38-45, 2003). However, such multipotent "embryonic-
like" stem cells
cannot be identified and isolated using the known markers.
The present invention provides methods of identifying, characterizing and
separating stem cells
having characteristics of embryonic stem (ES) cells for diagnostic, therapy
and tissue engineering.
In particular, the present invention provides methods of identifying,
selecting and separating
embryonic stem cells or fetal cells from maternal blood and to reagents for
use in prenatal diagnosis
and tissue engineering methods. The present invention provides for the first
time a specific
marker/binder/binding agent that can be used for identification, separation
and characterization of
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valuable stem cells from tissues and organs, overcoming the ethical and
logistical difficulties in the
currently available methods for obtaining embryonic stem cells.
The present invention overcomes the limitations of known binders/markers for
identification and
separation of embryonic or fetal stem cells by disclosing a very specific type
of marker/binder,
which does not react with differentiated somatic maternal cell types. In other
aspect of the
invention, a specific binder/marker/binding agent is provided which does not
react, i.e. is not
expressed on feeder cells, thus enabling positive selection of feeder cells
and negative selection of
stem cells.
By way of exemplification, the binder to Formulas according to the invention
are now disclosed as
useful for identifying, selecting and isolating pluripotent or multipotent
stem cells including
embryonic and embryonic type stem cells, which have the capability of
differentiating into varied
cell lineages.
According to one aspect of the present invention a novel method for
identifying pluripotent or
multipotent stem cells in peripheral blood and other organs is disclosed.
According to this aspect an
embryonic stem cell binder/marker is selected based on its selective
expression in stem cells and/or
germ stem cells and its absence in differentiated somatic cells and/or feeder
cells. Thus, glycan
structures expressed in stem cells are used according to the present invention
as selective
binders/markers for isolation of pluripotent or multipotent stem cells from
blood, tissue and organs.
Preferably the blood cells and tissue samples are of mammalian origin, more
preferably human
origin.
According to a specific embodiment the present invention provides a method for
identifying a
selective embryonic stem cell binder/marker comprising the steps of:
A method for identifying a selective stem cell binder to a glycan structure of
Formula (I) which
comprises:
i. selecting a glycan structure exhibiting specific expression in/on stem
cells and absence of
expression in/on feeder cells and/or differentiated somatic cells; ii. and
confirming the binding of
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binder to the glycan structure in/on stem cells.
By way of a non-limiting example, embryonic type, stem cells selected using
the binder may be
used in regenerating the hematopoietic or other tissue system of a host
deficient in any class of stem
cells. A host that is diseased can be treated by removal of bone marrow,
isolation of stem cells and
treatment with drugs or irradiation prior to re-engraftment of stem cells. The
novel markers of the
present invention may be used for identifying and isolating various embryonic
type stem cells;
detecting and evaluating growth factors relevant to stem cell self-
regeneration; the development of
stem cell lineages; and assaying for factors associated with stem cell
development.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Portrait of the hESC N-glycome. A. Mass spectrometric profiling of
the most abundant
50 neutral N-glycans (A) and 50 sialylated N-glycans (B) of the four hESC
lines (blue columns /
left), four EB samples (middle columns), and four stage 3 differentiated cell
samples (light
columns / right). The columns indicate the mean abundance of each glycan
signal (% of the total
glycan signals). Proposed N-glycan monosaccharide compositions are indicated
on the x-axis: S:
NeuAc, H: Hex, N: HexNAc, F: dHex, Ac: acetyl. The mass spectrometric glycan
profile was
rearranged and the glycan signals grouped in the main N-glycan structure
classes. Glycan signals in
the group `Other' are marked with m/z ratio of their [M+Na]+ (left panel) or
[M-H]- ions (right
panel). The isolated N-glycan fractions of hESC were structurally analyzed by
proton NMR
spectroscopy to characterize the major N-glycan core and backbone structures,
and specific
exoglycosidase digestions with a-mannosidase (Jack beans), a1,2-and 0,3/4-
fucosidases (X.
manihotis/recombinant), (31,4-galactosidase (S. pneumoniae), and neuraminidase
(A. ureafaciens) to
characterize the non-reducing terminal epitopes. Structures proposed for the
major N-glycan signals
are indicated by schematic drawings in the bar diagram. The major sialylated N-
glycan structures
are based on the trimannosyl core with or without core fucosylation as
demonstrated in the NMR
analysis. Galactose linkages or branch specificity of the antennae are not
specified in the present
data. The Lewis x antigen was detected in the same cells by monoclonal
antibody staining (not
shown).
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Figure 2. Mass spectrometric profiling of human embryonic stem cell and
differentiated cell N-glycans. A.
Neutral N-glycans and B. 50 most abundant sialylated N-glycans of the four
hESC lines (blue columns),
embryoid bodies derived from FES 29 and FES 30 hESC lines (EB, red columns),
and stage 3 differentiated
cells derived from FES 29 (st.3, white columns). The columns indicate the mean
abundance of each glycan
signal (% of the total detected glycan signals). Error bars indicate the range
of detected signal intensities.
Proposed monosaccharide compositions are indicated on the x-axis. H. hexose,
N. N-acetylhexosamine, F:
deoxyhexose, S: N-acetylneuraminic acid, G: N-glycolylneuraminic acid.
Figure 3. A. Classification rules for human N-glycan biosynthetic groups. The
minimal structures of each
biosynthetic group (solid lines) form the basis for the classification rules.
Variation of the basic structures by
additional monosaccharide units (dashed lines) generates complexity to stem
cell glycosylation as revealed in
the present study. H: hexose, N: N-acetylhexosamine, F: deoxyhexose, S: N-
acetylneuraminic acid. B.
Diagram showing relative differences in N-glycan classes between hESC and
stage 3 differentiated cells
(st.3). Although the major N-glycan classes are expressed in both hESC and the
differentiated cell types,
their relative proportions are changed during hESC differentiation. Complex
fucosylation (F>2) of sialylated
N-glycans as well as high-mannose type and complex-type N-glycans were
identified as the major hESC-
associated N-glycosylation features. In contrast, fucosylation as such (F>1)
was not similarly specific.
Hybrid-type or monoantennary, low-mannose type, and terminal N-
acetylhexosamine (N>H>2 or N-H>5)
type N-glycans were associated with differentiated cells. The relative
differences were calculated according
to Equation 2 from the N-glycan profiles (Supplementary Table S5). Schematic
examples of glycan
structures included in each glycan class are inserted in the diagram. Glycan
symbols: ^, N-acetyl-D-
glucosamine; 0, D-mannose; 0, D-galactose; *, N-acetylneuraminic acid; 0, L-
fucose; ^, N-acetyl-D-
galactosamine.
Figure 4. The major N-glycan structures in hESC N-glycome were determined by
MALDI-TOF mass
spectrometry combined with exoglycosidase digestion and proton NMR
spectroscopy. A, High-mannose type
N-glycans with five to nine mannose residues dominated the neutral N-glycan
fraction. B, In the sialylated
N-glycan fraction, the most abundant components were biantennary complex-type
N-glycans with either
a2,3 or a2,6-sialylated type 11 N-acetyllactosamine antennae and with or
without core al,6-fucosylation.
Glycan symbols: see legend of Figure 3; lines indicate glycosidic linkages
between monosaccharide residues;
dashed lines indicate the presence of multiple structures; --+Asn indicates
site of linkage to glycoprotein.
Figure 5. Statistical discrimination analysis of the four hESC lines, embryoid
bodies derived from FES 29
and FES 30 hESC lines (EB), and stage 3 differentiated cells derived from FES
29 (st.3). The calculation of
the glycan score is detailed in the Supplementary data.
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Figure 6. Lectin staining of hESC colonies grown on mouse feeder cell layers,
with A, Maackia
amurensis agglutinin (MAA) that recognizes a2,3-sialylated glycans, and with
B, Pisum sativum
agglutinin (PSA) that recognizes N-glycan core residues. PSA recognized hESC
only after cell
permeabilization (data not shown). Mouse fibroblasts had complementary
staining patterns with
both lectins, indicating that their surface glycans are clearly different from
hESC. C, The results
indicate that mannosylated N-glycans are localized primarily in the
intracellular compartments in
hESC, whereas a2,3-sialylated glycans occur on the cell surface.
Figure 7. 50 most abundant signals from the neutral N-glycome of human
embryonic stem cells.
Figure 8. Hybrid and complex N-glycans picked from the 50 most abundant
signals from the neutral
N-glycome of human embryonic stem cells.
Figure 9. 50 most abundant signals from the acidic N-glycome of human
embryonic stem cells.
Figure 10. (A) Hybrid N-glycans of human embryonic stem cells and changes in
their relative
abundance during differentiation. (B) Enlargement of the X-axis of (A).
Figure 11. High mannose N-glycans (Man > 5) of human embryonic stem cells and
changes in their
relative abundance during differentiation.
Figure 12. "Low mannose" N-glycans (Man 1-4) of human embryonic stem cells and
changes in
their relative abundance during differentiation.
Figure 13. (A) Fucosylated N-glycans of human embryonic stem cells and changes
in their relative
abundance during differentiation. (B) Enlargement of the X-axis of (A).
Figure 14. (A) "Complexly fucosylated" (Fuc > 2) N-glycans of human embryonic
stem cells and
changes in their relative abundance during differentiation. (B) Enlargement of
the X-axis of (A).
Figure 15. Sulfated N-glycans of human embryonic stem cells and changes in
their relative
abundance during differentiation.
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Figure 16. Large N-glycans (H>7, N>6) of human embryonic stem cells and
changes in their
relative abundance during differentiation.
Figure 17. Portrait of the hESC N-glycome. MALDI-TOF mass spectrometric
profiling of the
most abundant 50 neutral N-glycans (A.) and 50 sialylated N-glycans (B.) of
the four hESC lines
FES 21, 22, 29, and 30 (black columns), four EB samples (gray columns), and
four st.3
differentiated cell samples (white columns) derived from the four hESC lines,
respectively. The
colunms indicate the mean abundance of each glycan signal (% of the total
glycan signals). The
observed m/z values for either [M+Na]+ or [M-H]- ions for the neutral and
sialylated N-glycan
fractions, respectively, are indicated on the x-axis. Proposed monosaccharide
compositions and N-
glycan types are presented in Table 21.
Figure 18. Detection of hESC glycans by structure-specific reagents. To study
the localization of
the detected glycan components in hESC, stem cell colonies grown on mouse
feeder cell layers
were labeled by fluoresceinated glycan-specific reagents selected based on the
analysis results. A.
The hESC surfaces were stained by Maackia amurensis agglutinin (MAA),
indicating that a2,3-
sialylated glycans are abundant on hESC but not on feeder cells (MEF, mouse
feeder cells). B. In
contrast, the hESC cell surfaces were not stained by Pisum sativum agglutinin
(PSA) that
recognized mouse feeder cells, indicating that a-mannosylated glycans are not
abundant on hESC
surfaces but are present on mouse feeder cells. C. Addition of 3'-
sialyllactose blocks MAA binding
, and D. addition of D-mannose blocks PSA binding.
Figure 19. hESC-associated glycan signals selected from the 50 most abundant
sialylated N-glycan
signals of the analyzed hESC, EB, and st.3 samples (data taken from Fig. 1.B).
Figure 20. Differentiated cell associated glycan signals selected from the 50
most abundant
sialylated N-glycan signals of the analyzed hESC, EB, and st.3 samples (data
taken from Fig. 17.B).
Figure 21. A) Baboon polyclonal anti-Gala3Gal antibody staining of mouse
fibroblast feeder cells
(left) showing absence of staining in hESC colony (right). B) UEA (Ulex
Europaeus) lectin staining
of stage 3 human embryonic stem cells. FES 30 line.
Figure 22. A) UEA lectin staining of FES22 human embryonic stem cells
(pluripotent,
undifferentiated). B) UEA staining of FES30 human embryonic stem cells
(pluripotent,
undifferentiated).
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Figure 23. A) RCA lectin staining of FES22 human embryonic stem cells
(pluripotent,
undifferentiated). B) WFA lectin staining of FES30 human embryonic stem cells
(pluripotent,
undifferentiated).
Figure 24. A) PWA lectin staining of FES30 human embryonic stem cells
(pluripotent,
undifferentiated). B) PNA lectin staining of FES30 human embryonic stem cells
(pluripotent,
undifferentiated).
Figure 25. A) GF 284 immunostaining of FES30 human embryonic stem cell line.
Immunostaining
is seen in the edges of colonies in cells of early differentiation (lOx
magnification). Mouse feeder
cells do not stain. B) Detail of GF284 as seen in 40x magnification. This
antibody is suitable for
detecting a subset of hESC lineage.
Figure 26. A) GF 287 immunostaining of FES30 human embryonic stem cell line.
Immunostaining
is seen throughout the colonies (lOx magnification). Mouse feeder cells do not
stain. B) Detail of
GF287 as seen in 40x magnification. This antibody is suitable for detecting
undifferentiated,
pluripotent stem cells.
Figure 27. A) GF 288 immunostaining of FES30 human embryonic stem cells.
Immunostaining is
seen mostly in the edges of colonies in cells of early differentiation (lOx
magnification). Mouse
feeder cells do not stain. B) Detail of GF288 as seen in 40x magnification.
This antibody is suitable
for detecting a subset of hESC lineage
Figure 28. The canonical means of the first discriminant analysis for neutral
hESC, EB and st3.
Root 1 is represented on the x-axis and Root 2 on the y-axis. From the figure
we can see that the
means are further differentiated on the x-axis and therefore we use Root 1 to
determine the function.
Figure 29. The canonical means of the second minimal discriminant analysis for
neutral glycans
from hESC, EB and st3 (5 masses). Root 1 is represented on the x-axis and Root
2 on the y-axis.
Figure 30. The canonical means of the first minimal discriminant analysis for
neutral glycans from
hESC, EB and st3 (4 masses). Root 1 is represented on the x-axis and Root 2 on
the y-axis.
Figure 31. Lectin FACS of hESCs. hESCs were detached with EDTA, washed with
FCS-PBS.
FES30 cells were double staining with SSEA-3+.
Figure 32. FACS analysis using various antibodies. The cells were detached
with EDTA and
washed with buffer containing FCS.
DESCRIPTION OF THE INVENTION
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Related data and specification was presented in PCT Fl 2006/050336, for US
proceedings and when
relevant for other countries the applications are included as reference.
The present invention revealed novel stem cell specific glycans, with specific
monosaccharide
compositions and associated with differentiation status of stem cells and/or
several types of stem
cells and/or the differentiation levels of one stem cell type and/or lineage
specific differences
between stem cell lines.
The present invention is directed to human embryonic type stem cells and stem
cells and tissue
precursors differentiated thereof. It is realized that ethical considerations
may restrict patenting of
actual embryonic stem cells derived from human embryos, but there is numerous
technologies to
produce equivalent materials with less or no ethical concerns involved.
Furthermore non destructive
analysis of stem cells should not involve ethical problems.
Preferred target cell populations and types for analysis according to the
invention
Human embryonic type stem cells
Under broadest embodiment the present invention is directed to all types of
human embryonic type
stem cells, meaning fresh and cultured human embryonic type stem cells.
The stem cells according to the invention do not include traditional cancer
cell lines, which may
differentiate to resemble natural cells, but represent non-natural
development, which is typically due
to chromosomal alteration or viral transfection. It is realized that the data
from embryonal
carcinomas (EC) and EC cell lines is not relevant for embryonic stem cells.
The embryonic stem cells include all types of non-malignant embryonic
multipotent or totipotent
cells capable of differentiating to other cell types. The embryonic stem cells
have special capacity
stay as stem cells after cell division, the self-reneval capacity. The
preferred differentiated
derivatives of embryonic stem cells includes embryonic bodies, also referred
as stage 2
differentiated embryonic stem cells and stage three differentiated embryonic
stem cells. In a
preferred embodiment the the stage 3 embryonic stem cells have at least
partial characteristics of
specific tissue or more preferably characteristics of a specific tissue stem
cells.
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Under the broadest embodiment for the human stem cells, the present invention
describes novel
special glycan profiles and novel analytics, reagents and other methods
directed to the glycan
profiles. The invention shows special differences in cell populations with
regard to the novel glycan
profiles of human stem cells.
The present invention is further directed to the novel structures and related
inventions with regard to
the preferred cell populations according to the invention. The present
invention is further directed to
specific glycan structures, especially terminal epitopes, with regard to
specific preferred cell
population for which the structures are new.
Embryonic type cell populations
The present invention is specifically directed to methods directed to
embryonic type or "embryonic
like" cell populations, preferably when the use does not involve commercial or
industrial use of
human embryos and/or involve destruction of human embryos. The invention is
under a specific
embodiment directed to use of embryonic cells and embryo derived materials
such as embryonic
stem cells, whenever or wherever it is legally acceptable. It is realized that
the legislation varies
between countries and regions. The inventors reserve possibility to disclaim
legally restricted types
of embryonic stem cells.
The present invention is further directed to use of embryonic-related,
discarded or spontaneously
damaged material, which would not be viable as human embryo and cannot be
considered as a
human embryo. In yet another embodiment the present invention is directed to
use of accidentally
damaged embryonic material, which would not be viable as human embryo and
cannot be
considered as human embryo. Gene technology and embryonic biopsy based methods
producing ES
cells from embryos without damging the embryo to produce embryonic or
embryonic type stem
cells are expected to produce ethically acceptable or more cells.
In a preferred embodiment the invention is directed to embryonic type stem
cells, which are
produced from other cell types by programming the cells to undifferentiated
status corresponding to
embryonic stem cells or cells corresponding to the preferred differentiated
variants of the ES cells.
The invention is further directed to cell materials equivalent to the cell
materials according to the
invention. It is further realized that functionally and even biologically
similar cells may be obtained
by artificial methods including cloning technologies.
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N-glycan structures and compositions associated with differentiation of stem
cells
The invention revealed specific glycan monosaccharide compositions and
corresponding structures,
which associated with
i) non-differentiated human embryonic stem cells, hESCs (stage 1) or
ii) stage 2 (embryoid bodies) and/or
iii) stage 3 differentiated cells differentiated from the hESCs.
It is realized that the structures revealed are useful for the
characterization of the cells at different
stages of development. The invention is directed to the use of the structures
as markers for
differentiation of embryonic stem cells. The invention is further directed to
the use of the specific
glycans as markers enriched or increased at specific level of differentiation
for the analysis of the
cells at specific differentiation level.
Glycan structures and compositions are associated with individual specific
differences
between stem cell lines or batches.
The invention further revelead that specific glycan types are presented in the
embryonic stem cell
preparations on a specific differentiation stage in varying manner. It is
realized that such
individually varying glycans are useful for characterization of individual
stem cell lines and
batches. The specific structures of a individual cell preparation are useful
for comparison and
standardization of stem cell lines and cells prepared thereof.
The specific structures of a individual cell preparation are used for
characterization of usefulness of
specific stem cell line or batch or preparation for stem cell therapy in a
patient, who may have
antibodies or cell mediated immune defence recognizing the individually
varying glycans.
The invention is especially directed to analysis of glycans with large and
moderate variations as
described in example 3.
Recognition of multiple structures
The invention revealed multiple glycan structures and corresponding mass
spectrometric signals,
which are characteristic for the stem cell populations according to the
invention. In a preferred
embodiment the invention is directed to recognition of specific combinations
glycans such as
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whole glycans and/or corresponding signals, such as mass spectrometric signals
and/or specific
structural epitopes, preferably non-reducing end terminal glycans structures.
It is realized that certain combination of structures are useful for detection
because the change of
structures can be correlated with the status of the cell, in a preferred
embodiment the differentiation
status of the cells is correlated with the glycans. The invention specifically
revealed glycans
changing during the differentiation of the cells. It was revealed that certain
glycan structures are
increased and others decreased during differentiation of cells. The invention
is directed to use of
combinations of structures changing similaliry during differentiation and/or
structures changing
differently (at least one decreasing and at least one decreasing).
Analysis methods by mass spectrometry or specific bindin2 real!ents
The invention is specifically directed to the recognition of the terminal
structures by either specific
binder reagents and/or by mass spectrometric profiling of the glycan
structures.
In a preferred embodiment the invention is directed to the recognition of the
structures and/or
compositions based on mass spectrometric signals corresponding to the
structures.
The preferred binder reagents are directed to characteristic epitopes of the
structures such as
terminal epitopes and/or characteristic branching epitopes, such as
monoantennary structures
comprising a Mana-branch or not comprising a Mana-branch.
The preferred binder is an antibody, more preferably a monoclonal antibody.
In a preferred embodiment the invention is directed to a monoclonal antibody
specifically
recognizing at least one of the terminal epitope structures according to the
invention.
Recognition of preferred terminal epitopes
The invention is in a preferred embodiment directed to the analysis of the
stem cells by specific
antibodies and other binding reagents recognizing preferred structural
epitopes according to the
invention.
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The preferred structural epitopes includes non-reducing end terminal
Gal/Ga1NAc(33/4- epitope
comprising structures and sialyated and/or fucosylated derivatives thereof The
invention is directed
to recognition of at at least one N-acetylactos
Non-reducing end terminal Gal(NAc )beta structures
Terminal Galactose epitopes including
i) terminal N-acetyllactosamines Gal(33G1cNAc and/or Gal(34GlcNAc, and
fucosylated
branched variants thereof such as Lewis a[Gal(33(Fuc(x4)GIcNAc] and
Lewis x [Gal(34(Fuca3)G1cNAc]
ii) 0-glycan core structures including Gal(33GaLNAca in linear core I epitope
and/or
branched Gal(33(R-G1cNAc(36)GalNAca,
iii) Glycolipid structures with terminal Gal(33Ga1NAc(3 -structures
Terminal Ga1NAc epitopes including
i) terminal di-N-acetyllactosediamine Ga1NAc(34G1cNAc (LacdiNAc), and
a3fucosylated derivative thereof, LexNAc [Ga1NAc(34(Fuca3)G1cNAc]
ii) Glycolipid structures with terminal Ga1NAc(33Gal -structures
Sialylated non-reducing end terminal Gal(NAc)beta structures
The preferred terminal sialylated Gal(NAc) epitopes including,
The preferred sialic acid is (SA) such Neu5Ac or Neu5Gc.
i) terminal sialyl-N-acetyllactosamines SAa3/6Ga1(33G1cNAc and/or
SAa3/6Ga1(34G1cNAc, and fucosylated branched variants thereof such as sialyl-
Lewis a
[SAa3Gal(33(Fuca4)G1cNAc] and sialyl- Lewis x [SAa3Ga1(34(Fuc(x3)G1cNAc]
ii) sialylated 0-glycan core structures including SAa3Gal(33Ga1NAca in linear
core I
epitope or disialyl-structures SAa3Ga1(33(SAa6)Ga1NAca, and/or branched
SA(x3Ga1(33(R-G1cNAc(36)Ga1NAca,
iii) Glycolipid structures with terminal SAa3Gal(33GalNAc(3 -structures and
disialostructures SAa3Ga1(33(SA(x6)Ga1NAc(3, disialosyl-Tn).
Terminal sialylated GaLNAc epitopes including sialylated GaLNAc(33/4-
structures
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i) terminal sialyl di-N-acetyllactosediamine SAaGalNAc(34GlcNAc, more
preferably
SA(x6Ga1NAc(34G1cNAc
Fucosylated non-reducing end terminal Galbeta structures
The position 2 of galetose carrying N-acetylgroup in Ga1NAc can be fucosylated
to a preferred
strcture group with similarity to the terminal Ga1NAc structures The preferred
terminal fucosylated
Gal epitopes includes,
i) terminal fucoslyl-N-acetyllactosamines Fuca2Gal(33G1cNAc and/or
Fuca2Gal04G1cNAc, and fucosylated branched variants thereof such as Lewis b
[Fuca2Ga1(33(Fuc(x4)G1cNAc] and Lewis y [Fuca2Gal(34(Fuca3)G1cNAc]
ii) fucosylated 0-glycan core structures including Fuca2Ga1(33Ga1NAca in
linear core I
epitope and/or branched Fuca2Ga1(33(R-G1cNAc(36)Ga1NAc(x,
iii) Glycolipid structures with terminal Fuca2Ga1(33Ga1NAc(3 -structures.
Terminal structural epitopes
We have previously revealed glycome compositions of human glycomes,
here we provide structural terminal epitopes useful for the cahracterization
of stem cell glycomes,
especially by specific binders.
The examples of characteristic altering terminal structures includes
expression of competing
terminal epitopes created as modification of key homologous core Gal(3-
epitopes, with either the
same monosaccharides with difference in linkage position Gal(33G1cNAc, and
analogue with either
the same monosaccharides with difference in linkage position Gal(34G1cNAc; or
the with the same
linkage but 4-position epimeric backbone Gal(33Ga1NAc. These can be presented
by specific core
structures modifying the biological recognition and function of the
structures. Another common
feature is that the similar Gal(3-structures are expressed both as protein
linked (0- and N-glycan)
and lipid linked (glycolipid structures). As an alternative for a2-
fucosylation the terminal Gal may
comprise NAc group on the same 2 position as the fucose. This leads to
homologous epitopes
GaLNAc(34GlcNAc and yet related Ga1NAc(33Ga1-structure on characteristic
special glycolipid
according to the invention.
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The invention is directed to novel terminal disaccharide and derivative
epitopes from human stem
cells, preferably from human embryonic type stem cells. It should realized
that glycosylations are
species, cell and tissue specific and results from cancer cells usually differ
dramatically from
normal cells, thus the vast and varying glycosylation data obtained from human
embryonal
carcinomas are not actually relevant or obvious to human embryonic stem cells
(unless accidentally
appeared similar). Additionally the exact differentiation level of
teratocarcinomas cannot be known,
so comparision of terminal epitope under specific modification machinery
cannot be known. The
terminal structures by specific binding molecules including glycosidases and
antibodies and
chemical analysis of the structures.
The present invention reveals group of terminal Gal(NAc)(31-3/4Hex(NAc)
structures, which carry
similar modifications by specific fucosylation/NAc-modification, and
sialylation on corresponding
positions of the terminal disaccharide epitopes. It is realized that the
terminal structures are
regulated by genetically controlled homologous family of fucosyltransferases
and sialyltransferases.
The regulation creates a characteristic structural patterns for communication
between cells and
recognition by other specific binder to be used for analysis of the cells. The
key epitopes are
presented in the TABLE 21. The data reveals characteristic patterns of the
terminal epitopes for
each types of cells, such as for example expression on hESC-cells generally
much Fuca-structures
such as Fuca2-structures on type 1 lactosamine (Gal03G1cNAc), similarily (33-
linked core I
Gal(33GlcNAca, and type 4 structure which is present on specific type of
glycolipids and
expression of a3-fucosylated structures, while a6-sialic on type II N-
acetylalactosamine appear on
N-glycans of embryoid bodies and st3 embryonic stem cells. E.g. terminal type
lactosamine and
poly-lactosamines differentiate stem cells with different status such as
differentiation status. The
terminal Gal(3-information is preferably combined with information about
information about other
preferred terminal structures such as sialyalted and/or fucosylated
structures.
The invention is directed especially to high specificity binding molecules
such as monoclonal
antibodies for the recognition of the structures.
The structures can be presented by Formula Tl. the formula describes first
monosaccharide residue
on left, which is a(3-D-galactopyranosyl structure linked to either 3 or 4-
position of
the a- or (3-D-(2-deoxy-2-acetamido)galactopyranosyl structure, when R5 is OH,
or (3-D-(2-deoxy-2-acetamido)glucopyranosyl, when R4 comprises 0-. The
unspecified
stereochemistry of the reducing end in formulas Tl and T2 is indicated
additionally (in claims) with
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curved line. The sialic acid residues can be linked to 3 or 6-position of Gal
or 6-position of G1cNAc
and fucose residues to position 2 of Gal or 3- or 4-position of G1cNAc or
position 3 of Glc.
The invention is directed to Galactosyl-globoside type structures comprising
terminal Fuca2-
revealed as novel terminal epitope Fuca2Ga1(33Ga1NAc(3 or Gal(33GaLNAc(3Gala3-
comprising
isoglobotructures revealed from the embryonic type cells.
Formula T1
R5 R6
OH Ri
O
O Ra ~
O~ O [xJ ~/
RZ R3 R7
m
wherein
X is linkage position
Ri, R2, and R6 are OH or glycosidically linked monosaccharide residue Sialic
acid, preferably
Neu5Aca2 or Neu5Gc a2, most preferably Neu5Aca2 or
R3, is OH or glycosidically linked monosaccharide residue Fucal (L-fucose) or
N-acetyl (N-
acetamido, NCOCH3);
R4, is H, OH or glycosidically linked monosaccharide residue Fucal (L-fucose),
R5 is OH, when R4 is H, and R5 is H, when R4 is not H;
R7 is N-acetyl or OH
X is natural oligosaccharide backbone structure from the cells, preferably N-
glycan, 0-glycan or
glycolipid structure; or X is nothing, when n is 0,
Y is linker group preferably oxygen for 0-glycans and 0-linked terminal
oligosaccharides and
glycolipids and N for N-glycans or nothing when n is 0;
Z is the carrier structure, preferably natural carrier produced by the cells,
such as protein or lipid,
which is preferably a ceramide or branched glycan core structure on the
carrier or H;
The arch indicates that the linkage from the galactopyranosyl is either to
position 3 or to position 4
of the residue on the left and that the R4 structure is in the other position
4 or 3;
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n is an integer 0 or 1, and m is an integer from 1 to 1000, preferably 1 to
100, and most preferably 1
to 10 (the number of the glycans on the carrier),
With the provisions that one of R2 and R3 is OH or R3 is N-acetyl,
R6 is OH, when the first residue on left is linked to position 4 of the
residue on right:
X is not Gala4Gal04G1c, (the core structure of SSEA-3 or 4) or R3 is Fucosyl
R7 is preferably N-acetyl, when the first residue on left is linked to
position 3 of the residue on
right:
Preferred terminal (33-linked subgroup is represented
by Formula T2 indicating the situation, when the first residue on the left is
linked to the 3 position
with backbone structures Gal(NAc)03Ga1/G1cNAc.
OH R, R5 Re
O
O R4
o ~-O X y Z
RZ NH
Rs O~
CH3
m
Formula T2
Wherein the variables including Ri to R7
are as described for T1
Preferred terminal (34-linked subgroup is represented by the Formula 3
Formula T3
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OH R, OH
O O O
O X y z
R Rq
Rz
3 R7
m
Wherein the variables including Ri to R4 and R7
are as described for T1 with the provision that
R4, is OH or glycosidically linked monosaccharide residue Fucal (L-fucose),
Alternatively the epitope of the terminal structure can be represented by
Formulas T4 and T5
Core Gal(3-epitopes formula T4:
Gal(31-xHex(NAc)p,
x is linkage position 3 or 4,
and Hex is Gal or Glc
with provision
pis0orl
when x is linkage position 3, p is 1 and HexNAc is GIcNAc or Ga1NAc,
and when x is linkage position 4, Hex is Glc.
The core Gal(31-3/4 epitope is optionally substituted to hydroxyl
by one or two structures SAa or Fuca, preferably selected from the group
Gal linked SAa3 or SAa6 or Fuca2, and
Glc linked Fuca3 or G1cNAc linked Fuca3/4.
Formula T5
[Ma]mGal(31-x[Na]nHex(NAc)p,
wherein m, n and p are integers 0, or 1, independently
Hex is Gal or Glc,
X is linkage position
M and N are monosaccharide residues being
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independently nothing (free hydroxyl groups at the positions)
and/or
SA which is Sialic acid linked to 3-position of Gal or/and 6-position of
HexNAc
and/or
Fuc (L-fucose) residue linked to 2-position of Gal
and/or 3 or 4 position of HexNAc, when Gal is linked to the other position (4
or 3),
and HexNAc is G1cNAc, or 3-position of Glc when Gal is linked to the other
position (3),
with the provision that sum of m and n is 2
preferably m and n are 0 or 1, independently.
The exact structural details are essential for optimal recognition by specific
binding molecules
designed for the analysis and/or manipulation of the cells.
The terminal key Gal(3-epitopes are modified by the same modification
monosaccharides NeuX (X is 5 position modification Ac or Gc of sialic acid) or
Fuc,
with the same linkage type alfa( modifying the same hydroxyl-positions in both
structures.
NeuXa3, Fuca2 on the terminal Gal(3 of all the epitopes and
NeuXa6 modifying the terminal Gal(3 of Gal(34G1cNAc, or HexNAc, when linkage
is 6 competing
or Fuca modifying the free axial primary hydroxyl left in GIcNAc (there is no
free axial hydroxyl
in Ga1NAc-residue).
The preferred structures can be divided to preferred Gal(31-3 structures
analogously to T2,
Formula T6:
[Ma]mGal(31-3[Na]nHexNAc,
Wherein the variables are as described for T5.
The preferred structures can be divided to preferred Gal(31-4 structures
analogously to T4,
Formula T7:
[Ma]mGal(31-4[Na]nGlc(NAc)p,
Wherein the variables are as described for T5.
These are preferred type 11 N-acetyllactosamine structures and related
lactosylderivatives, in a
preferred embodiment p is 1 and the structures includes only type 2 N-
acetyllactosamines. The
invention revealed that the these are very useful for recognition of specific
subtypes of embryonic
type stem cells or differentiated variants thereof (tissue type specifically
differentiated embryonic
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stem cells or various stages of embryonic stem cells). It is notable that
various fucosyl- and or sialic
acid modification created characteristic pattern for the stem cell type.
Preferred type I and type II N-acetyllactosamine structures
The preferred structures can be divided to preferred type one (I) and type two
(II) N-
acetyllactosamine structures comrising oligosaccharide core sequence Gal(31-
3/4 G1cNAc structures
analogously to T4,
Formula T8:
[Ma]Gal(31-3/4[Na]nG1cNAc,
Wherein the variables are as described for T5.
The preferred structures can be divided to preferred Gal(31-3 structures
analogously to T8,
Formula T9:
[Ma]mGal(31-3[Na]nG1cNAc
Wherein the variables are as described for T5.
These are preferred type I N-acetyllactosamine structures. The invention
revealed that the these are
very useful for recognition of specific subtypes of the embryonic type stem
cells or differentiated
variants thereof (tissue type specifically differentiated embryonic type stem
cells or various stages
of embryonic stem cells). It is notable that various fucosyl- and or sialic
acid modification created
characteristic pattern for the stem cell type.
The preferred structures can be divided to preferred Gal(31-4G1cNAc core
sequence comprising
structures analogously to T8,
Formula T10:
[Ma]mGal(31-4[Na]nG1cNAc
Wherein the variables are as described for T5.
These are preferred type II N-acetyllactosamine structures. The invention
revealed that the these are
very useful for recognition of specific subtypes of embryonic type stem cells
or differentiated
variants thereof (tissue type specifically differentiated embryonic type stem
cells or various stages
of embryonic stem cells).
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It is notable that various fucosyl- and or sialic acid modificationally N-
acetyllactosamine structures
create especiaaly characteristic pattern for the stem cell type. The invention
is further directed to use
of combinations binder reagents recognizing at least two different type I and
type II
acetyllactosamines including at least one fucosylated or sialylated varient
and more preferably at
least two fucosylated variants or two sialylated variants
Preferred structures comprising terminal Fuca2/3/4-structures
The invention is further directed to use of combinations binder reagents
recognizing:
a) type I and type II acetyllactosamines and their fucosylated variants, and
in a preferred
embodiment
b) non-sialylated fucosylated and even more preferably
c) fucosylated type I and type II N-acetyllactosamine structures preferably
comprising Fuc(x2-
terminal and/or Fuca3/4-branch structure and even more preferably
d) fucosylated type I and type II N-acetyllactosamine structures preferably
comprising Fuca2-
terminal
for the methods according to the invention of various stem cells especially
embryonic type and
differentiated variants thereof.
Preferred subgroups of Fuca2-structures includes monofucosylated H type and H
type II structures,
and difucosylated Lewis b and Lewis y structures.
Preferred subgroups of Fuca3/4-structures includes monofucosylated Lewis a and
Lewis x
structures, sialylated sialyl-Lewis a and sialyl-Lewis x- structures and
difucosylated Lewis b and
Lewis y structures.
Preferred type II N-acetyllactosamine subgroups of Fuca3-structures includes
monofucosylated
Lewis x structures, and sialyl-Lewis x- structures and Lewis y structures.
Preferred type I N-acetyllactosamine subgroups of Fuca4-structures includes
monofucosylated
Lewis a sialyl-Lewis a and difucosylated Lewis b structures.
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The invention is further directed to use of at least two differently
fucosylated type one and or and
two N-acetyllactosamine structures preferably selected from the group
monofucosylated or at least
two difucosylated, or at least one monofucosylated and one difucosylated
structures.
The invention is further directed to use of combinations binder reagents
recognizing fucosylated
type I and type II N-acetyllactosamine structures together with binders
recognizing other terminal
structures comprising Fuca2/3/4-comprising structures, preferably Fuca2-
terminal structures,
preferably comprising Fuca2Ga1(33Ga1NAc-terminal, more preferably
Fuca2Ga1(33GaLNAca/(3 and
in especially preferred embodiment antibodies recognizing Fuca2Gal(33Ga1NAc(3-
preferably in
terminal structure of Globo- or isoglobotype structures.
Preferred Globo- and ganglio core type- structures
The invention is further directed to general formula comprising globo and
gangliotype Glycan core
structures according to formula
Formula Tl l
[M]mGal(31-x[Na]nHex(NAc)p, wherein m, n and p are integers 0, or 1,
independently
Hex is Gal or Glc, X is linkage position;
M and N are monosaccharide residues being
independently nothing (free hydroxyl groups at the positions)
and/or
SAa which is Sialic acid linked to 3-position of Gal or/and 6-position of
HexNAc
Gala linked to 3 or 4-position of Gal, or
GaLNAc(3linked to 4-position of Gal and/or
Fuc (L-fucose) residue linked to 2-position of Gal
and/or 3 or 4 position of HexNAc, when Gal is linked to the other position (4
or 3),
and HexNAc is G1cNAc, or 3-position of Glc when Gal is linked to the other
position (3),
with the provision that sum of m and n is 2
preferably m and n are 0 or 1, independently, and
with the provision that when M is Gala then there is no sialic acid linked to
Ga1(31, and
n is 0 and preferably x is 4.
with the provision that when M is Ga1NAc(3, then there is no sialic acid a6-
linked to Ga1(31, and n is
0andxis4.
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The invention is further directed to general formula comprising globo and
gangliotype Glycan core
structures according to formula
Formula T12
[M] [SAa3]nGal(31-4G1c(NAc)p,
wherein n and p are integers 0, or 1, independently
M is Gala linked to 3 or 4-position of Gal, or GaLNAc(3 linked to 4-position
of Gal
and/or SAa is Sialic acid branch linked to 3-position of Gal
with the provision that when M is Gala then there is no sialic acid linked to
Gal(31 (n is 0).
The invention is further directed to general formula comprising globo and
gangliotype Glycan core
structures according to formula
Formula T13
[M][SAa]nGal(31-4G1c,
wherein n and p are integer 0, or 1, independently
M isGala linked to 3 or 4-position of Gal, or
GaLNAc(3linked to 4-position of Gal
and/or
SAa which is Sialic acid linked to 3-position of Gal
with the provision that when M is Gala then there is no sialic acid linked to
Gal(31 (
n is 0).
The invention is further directed to general formula comprising globo type
Glycan core structures
according to formula
Formula T14
Gala3/4Gal(31-4G1c.
The preferred Globo-type structures includes Gala3/4Ga1P1-4G1c,
Ga1NAc(33Gala3/4Ga1(34G1c,
Gala4Ga1(34Glc (globotriose, Gb3), Gala3Gal(34Glc (isoglobotriose),
Ga1NAc(33Gala4Ga1(34G1c
(globotetraose, Gb4 (or G14)), and Fuca2Ga1(33Ga1NAc(33Gala3/4Ga1(34G1c. or
when the binder is not used in context of non-differentiated embryonal stem
cells or the binder is
used together with another preferred binder according to the invention,
preferably an other globo-
type binder the preferred binder targets furhter includes
Gal(33GaLNAc(33Gala4Ga1(34G1c (SSEA-3 antigen) and/or
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NeuAca3Gal(33GalNAc(33Gala4Ga1(34G1c (SSEA-4 antigen) or terminal non-reducing
end di or
trisaccharide epitopes thereof.
The preferred globotetraosylceramide antibodies does not recognize non-
reducing end elongated
variants of Ga1NAc(33Gala4Ga1(34G1c. The antibody in the examples has such
specificity as
The invention is further directed to binders for specific epitopes of the
longer oligosaccharide
sequences including preferably NeuAca3Ga1(33GaLNAc, NeuAca3Ga1(33Ga1NAc(3,
NeuAca3Gal(33GalNAc(33Gala4Ga1 when these are not linked to glycolipids and
novel fucosylated
target structures:
Fuca2Ga1(33GalNAc(33Gala3/4Ga1,Fuca2Gal(33Ga1NAc(33Gala,
Fuca2Ga1(33Ga1NAc(33Ga1, Fuc
a2Gal(33Ga1NAc(33, and Fuca2Gal(33Ga1NAc.
The invention is further directed to general formula comprising globo and
gangliotype Glycan core
structures according to formula
Formula T15
[Ga1NAc(34][SAa]nGal(31-4G1c, wherein n and p are integer 0, or 1,
independently Ga1NAc(3 linked
to 4-position of Gal and/or SAa which is Sialic acid branch linked to 3-
position of Gal.
The preferred Ganglio-type structures includes Ga1NAc(34Ga1(31-4G1c,
Ga1NAc(34[SAa3]Gal(31-
4Glc, and Gal(33Ga1NAc(34[SAa3]Gal(31-4G1c.
The preferred binder target structures further include glycolipid and possible
glycoprotein
conjugates of of the preferred oligosaccharide sequences. The preferred
binders preferably
specifically recognizes at least di- or trisaccharide epitope
GalNAca-structures
The invention is further directed to recognition of peptide/protein linked
GaLNAca-structures
according to the Formula T16:[SAa6]mGalNAca[Ser/Thr]n-[Peptide]p,wherein m, n
and p are
integers 0 or 1, independently,
wherein SA is sialic acid preferably NeuAc,Ser/Thr indicates linking serine or
threonine residues,
Peptide indicates part of peptide sequence close to linking residue,
with the provisio that either m or n is 1.
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Ser/Thr and/or Peptide are optionally at least partiallt necessary for
recognition for the binding by
the binder. It is realized that when Peptide is included in the specificity,
the antibody have high
specificity involving part of a protein structure. The preferred antigen
sequences of sialyl-Tn:
SAa6GalNAca, SAa6GaLNAcaSer/Thr, and SAa6GalNAcaSer/Thr-Peptide and Tn-
antigen:
GaLNAcaSer/Thr, and Ga1NAc(xSer/Thr-Peptide. The invention is further directed
to the use of
combinations of the Ga1NAca-structures and combination of at least one Ga1NAca-
structure with
other preferred structures.
Combinations of preferred binder r~oups
The present invention is especially directed to combined use of at least
a)fucosylated, preferably (x2/3/4-fucosylated structures and/or b) globo-type
structures and/or c)
GaLNAca-type structures. It is realized that using a combination of binders
recognizing strctures
involving different biosynthesis and thus having characteristic binding
profile with a stem cell
population. More preferably at least one binder for a fucosylated structure
and and globostructures,
or fucosylated structure and GalNAca-type structure is used, most preferably
fucosylated structure
and globostructure are used.
Fucosylated and non-modified structures
The invention is further directed to the core disaccharide epitope structures
when the structures are
not modified by sialic acid (none of the R-groups according to the Formulas T1-
T3 or M or N in
formulas T4-T7 is not sialic acid.
The invention is in a preferred embodiment directed to structures, which
comprise at least one
fucose residue according to the invention. These structures are novel specific
fucosylated terminal
epitopes, useful for the analysis of stem cells according to the invention.
Preferably native stem
cells are analyzed.
The preferred fucosylated structures include novel a3/4fucosylated markers of
human stem cells
such as (SAa3)ooriGal(33/4(Fuc(x4/3)G1cNAc including Lewis x and and
sialylated variants thereof.
Among the structures comprising terminal Fuca 1-2 the invention revealed
especially useful novel
marker structures comprising Fuca2Gal(33Ga1NAca/(3 and
Fuca2Ga1(33(Fuca4)0or1G1cNAc(3, these
were found useful studying embryonic stem cells. A especially preferred
antibody/binder group
among this group is antibodies specific for Fuca2Gal(33G1cNAc(3, preferred for
high stem cell
specificty. Another preferred structural group includes Fuca2Gal comprising
glycolipids revealed
to form specific structural group, especially interesting structure is globo-H-
type structure and
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29
glycolipids with terminal Fuca2Ga1(33Ga1NAc(3, preferred with interesting
biosynthetic context to
earlier speculated stem cell markers.
Among the antibodies recognizing Fuca2Ga1(34G1cNAc(3 substantial variation in
binding was
revealed likely based on the carrier structures, the invention is especially
directed to antibodies
recognizing this type of structures, when the specificity of the antibody is
similar to the ones
binding to the embryonic stem cells as shown in Example 18 with fucose
recognizing antibodies.
The invention is preferably directed to antibodies recognizing
Fuca2Gal(34GlcNAc(3 on N-glycans,
revealed as common structural type in terminal epitope Table 21.In a separate
embodiment the
antibody of the non-binding clone is directed to the recognition of the feeder
cells.
The preferred non-modified structures includes Gal(34G1c, Gal(33G1cNAc,
Gal(33Ga1NAc,
Gal(34GlcNAc, Gal(33G1cNAc(3, Gal(33GalNAc(3/a, and Gal(34G1cNAc(3. These are
preferred novel
core markers characteristics for the various stem cells. The structure
Gal(33G1cNAc is especially
preferred as novel marker observable in hESC cells. Preferably the structure
is carried by a
glycolipid core structure according to the invention or it is present on an 0-
glycan. The non-
modified markers are preferred for the use in combination with at least one
fucosylated or/and
sialylated structure for analysis of cell status.
Additional preferred non-modified structures includes Ga1NAc(3-structures
includes terminal
LacdiNAc, Ga1NAc(34G1cNAc, preferred on N-glycans and Ga1NAc(33Ga1
GalNAc(33Ga1 present
in globoseries glycolipids as terminal of globotetraose structures.
Among these characteristic subgroup of Gal(NAc)(33-comprising Gal(33GlcNAc,
Gal(33GaLNAc,
Gal(33GlcNAc(3, Gal(33Ga1NAc(3/a, and GaLNAc(33Gal Ga1NAc(33Gal and
the characteristic subgroup of Gal(NAc)(34-comprising Gal(34G1c, Gal(34G1cNAc,
and
Gal(34G1cNAc are separately preferred.
Preferred sialylated structures
The preferred sialylated structures includes characteristic SAa3Ga1(3-
structures SAa3Gal(34G1c,
SAa3Gal(33G1cNAc, SAa3Gal(33GalNAc, SAa3Ga1(34G1cNAc, SAa3Ga1(33G1cNAc(3,
SAa3Gal(33GalNAc(3/a, and SAa3Ga1(34G1cNAc(3; and biosynthetically partially
competing
SAa6Gal(3-structures SAa6Ga1(34Glc, SAa6Ga1(34G1c(3; SAa6Gal(34GlcNAc and
SAa6Gal(34GlcNAc(3; and disialo structures SAa3Ga1(33(SAa6)Ga1NAc(3/a,
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The invention is preferably directed to specific subgroup of Gal(NAc)(33-
comprising
SAa3Gal03GlcNAc, SAa3Gal03Ga1NAc, SAa3Ga104G1cNAc, SAa3Ga1(33G1cNAc(3,
SAa3Gal(33GaLNAc(3/a and SAa3Ga1(33(SA(x6)GaINAc(3/a,and
Gal(NAc)(34-comprising sialylated structures. SAa3Ga1(34G1c, and
SAa3Ga1(34G1cNAc(3; and
SAa6Gal04G1c, SAa6Gal(34Glc(3; SAa6Ga1(34G1cNAc and SAa6Ga1(34G1cNAc(3
These are preferred novel regulated markers characteristics for the various
stem cells.
Use to~4ether with a terminal ManaMan-structure
The terminal non-modified or modified epitopes are in preferred embodiment
used together with at
least one ManaMan-structure. This is preferred because the structure is in
different N-glycan or
glycan subgroup than the other epitopes.
Core structures of the terminal epitopes
It is realized that the target epitope structures are most effectively
recognized on specific N-glycans,
0-glycan, or on glycolipid core structures.
Elongated epitopes - Next monosaccharide/structure on the reducing end of the
epitope
The invention is especially directed to optimized binders and production
thereof, when the binding
epitope of the binder includes the next linkage structure and even more
preferably at least part of
the next structure (monosaccharide or aminoacid for O-glycans or ceramide for
glycaolipid) on the
reducing side of the target epitope. The invention has revealed the core
structures for the terminal
epitopes as shown in the Examples and ones summarized in Table 21.
It is realized that antibodies with longer binding epitopes have higher
specificity and thus will
recognize that desired cells or cell derived components more effectively. In a
preferred embodiment
the antibodies for elongated epitopes are selected for effective analysis of
embryonic type stem
cells.
The invention is especially directed to the methods of antibody selection and
optionally further
purification of novel antibodies or other binders using the elongated epitopes
according to the
invention. The preferred selection is performed by contacting the glycan
structure (synthetic or
isolated natural glycan with the specific sequence) with a serum or an
antibody or an antibody
library, such as a phage display library. Data about these methods are well
known in the art and
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31
available from internet for example by searching pubmed-medical literature
database
(www.ncbi.nlm.nih.gov/entrez) or patents e.g. in espacenet (fi.espacenet.com)
.
The specific antibodies are especially preferred for the use of the optimized
recognition of the
glycan type specific terminal structures as shown in the Examples and ones
summarized in the
Table 21.
It is further realized that part of the antibodies according to the invention
and shown in the
examples have specificity for the elongated epitopes. The inventors found out
that for example
Lewis x epiotpe can be recognized on N-glycan by certain terminal Lewis x
specific antibodies, but
not so effectively or at all by antibodies recognizing Lewis x(31-3Ga1 present
on poly-N-
acetyllactosamines or neolactoseries glycolipids.
N-glycans
The invention is especially directed to recognition of terminal N-glycan
epitopes on biantennary N-
glycans. The preferred non-reducing end monosaccharide epitope for N-glycans
comprise (32Man
and its reducing end further elongated variants
(32Man, (32Mana, (32Mana3, and (32Mana6
The invention is especially directed to recognition of lewis x on N-glycan by
N-glycan Lewis x
specific antibody described by Ajit Varki and colleagues Glycobiology (2006)
Abstracts of
Glycobiology society meeting 2006 Los Angeles, with possible implication for
neuronal cells,
which are not directed (but disclaimed) with this type of antibody by the
present invention.
Invention is further directed to antibodies with speficity of type 2 N-
acetyllactosamine(32Man
recognizing biantennary N-glycan directed antibody as described in Ozawa H et
al (1997) Arch
Biochem Biophys 342, 48-57.
0-glycans, reducing end elongated epitopes
The invention is especially directed to recognition of terminal 0-glycan
epitopes as terminal core I
epitopes and as elongated variants of core I and core II 0-glycans.
The preferred non-reducing end monosaccharide epitope for 0-glycans comprise:
a)Core I epitopes linked to (xSer/Thr- [Peptide]o_i,
wherein Peptide indicates peptide which is either present or absent. The
invention is preferabl
b) Preferred core II-type epitopes
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32
RI (36[R2(33Gal(33]nGalNAcaSer/Thr, wherein n is = or 1 indicating possible
branch in the structure
and RI and R2 are preferred positions of the terminal epitopes, Rl is more
preferred
c) Elongated Core I epitope
(33Ga1 and its reducing end further elongated variants (33Ga1(33GaLNAca,
(33Ga1(33Ga1NAcaSer/Thr
0-glycan core I specific and ganglio/globotype core reducing end epitopes have
been described in
(Saito S et al. J Biol Chem (1994) 269, 5644-52), the invention is preferably
directed to similar
specific recognition of the epitopes according to the invention.
0-glycan core II sialyl-Lewis x specific antibody has nbeen described in
Walcheck B et al. Blood
(2002) 99, 4063-69.
Peptide specificity including antibodies for recognition of 0-glycans includes
mucin specific
antibodies further recognizing Ga1NAcalfa (Tn) or Ga1b3Ga1NAcalfa (T/TF)
structures (Hanisch F-
G et al (1995) cancer Res. 55, 4036-40; Karsten U et al. Glycobiology (2004)
14, 681-92;
Glycolipid core structures
The invention is furthermore directed to the recognition of the structures on
lipid structures. The
preferred lipid corestructures include:
a) (3Cer (ceramide) for Ga1(34G1c and its fucosyl or sialyl derivatives
b) (33/6Gal for type I and type II N-acetyllactosamines on lactosyl Cer-
glycolipids, preferred
elongated variants includes (33/6[R(36/3]nGal(3, (33/6[R(36/3]nGal(34 and
03/6[R(36/3]nGal(34G1c, which may be further banched by another lactosamine
residue
which may be partially recognized as larger epitope and n is 0 or 1 indicating
the branch,
and Rl and R2 are preferred positions of the terminal epitopes. Preferred
linear (non-
branched) common structures include (33Ga1, (33Ga1(3, (33Ga1(34 and
(33Ga1(34GIc
c) a3/4Ga1, for globoseries epitopes, and elongated variants a3/4Ga1(3,
a3/4Ga1(34G1c
preferred globoepitopes have elongated epitopes a4Ga1, a4Ga1(3, (AGa1(34G1c,
and
preferred isogloboepitopes have elongated epitopes a3Gal, a3Gal(3, a3Gal(34G1c
d) (34Ga1 for ganglio-series epitopes comprising , and preferred elongated
variants include
(34Ga1(3, and (34Ga1(34G1c
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33
0-glycan core specific and ganglio/globotype core reducing end epitopes have
been described in
(Saito S et al. J Biol Chem (1994) 269, 5644-52), the invention is preferably
directed to similar
specific recognition of the epitopes according to the invention.
Poly-N-acetyllactosamines
Poly-N-acetyllactosamine backbone structures on 0-glycans, N-glycans, or
glycolipids comprise
characteristic structures similar to lactosyl(cer) core structures on type I
(lactoseries) and type II
(neolacto) glycolipids, but terminal epitopes are linked to another type I or
type II N-
acetyllactosamine, which may from a branched structure. Preferred elongated
epitopes include:
(33/6Ga1 for type I and type II N-acetyllactosamines epitope, preferred
elongated variants includes
Rl(33/6[R2(36/3]nGal(3, R103/6[R2(36/3]nGal(33/4 and
Rl(33/6[R206/3]nGal(33/4G1cNAc, which
may be further banched by another lactosamine residue which may be partially
recognized as larger
epitope and n is 0 or 1 indicating the branch, and Rl and R2 are preferred
positions of the terminal
epitopes. Preferred linear (non-branched) common structures include (33Ga1,
(33Ga1(3, (33Ga1(34 and
(33Ga1(34G1cNAc.
Numerous antibodies are known for linear (i-antigen) and branched poly-N-
acetyllactosamines (I-
antigen), the invention is further directed to the use of the lectin PWA for
recognition of I-antigens.
The inventors revelealed that poly-N-acetyllactosamines are characteristic
structures for specific
types of human stem cells. Another preferred binding regent, enzyme endo-beta-
galactosidase was
used for characterization poly-N-acetyllactosamines on glycolipids and on
glycoprotein of the stem
cells. The enzyme revealed characteristic expression of both linear and
branched poly-N-
acetyllactosamine, which further comprised specific terminal modifications
such as fucosylation
and/or sialylation according to the invention on specific types of stem cells.
Combinations of elongated core epitopes
It is realized that stronger labeling may be obtained if the same terminal
epitope is recognized by
antibody binding to target structure present on two or three of the major
carrier types 0-glycans, N-
glycans and glycolipids. It is further realized that in context of such use
the terminal epitope maust
be specific enough in comparision to the epitopes present on possible
contaminating cells or cell
matrials. It is further realized that there is highly terminally specific
antibodies, which allow binding
to on several elongation structures.
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34
The invention revealed each elongated binder type useful in context of stem
cells. Thus the
invention is directed to the binders recognizing the terminal structure on one
or several of the
elongating structures according to the invention
Preferred group of monosaccharide elongation structures
The invention is directed to use of binders with elongated specificity, when
the binders recognize or
is able to bind at least one reducing end elongation monosaccharide epitope
according to the
formula
AxHex(NAc)n, wherein A is anomeric structure alfa or beta,X is linkage
position 2, 3,4, or 6
And Hex is hexopyranosyl residue Gal, or Man, and n is integer being 0 or 1,
with the provisions
that when n is 1 then AxHexNAc is 06GaLNAc, when Hex is Man, then AxHex is
(32Man, and
when Hex is Gal, then AxHex is (33Ga1 or (36Ga1.
Beside the monosaccharide elongation structures aSer/Thr are preferred
reducing end elongation
structures for reducing end Ga1NAc-comprising 0-glycans and (3Cer is preferred
for lactosyl
comprising glycolipid epitopes.
The invention is directed to the preferred terminal epitopes according to the
invention comprising
the preferred reducing end elongation of the N-acetyllactosamine epitomes
described in Formulas
T 1-T 11, referred as T 1 E-T 11 E in elongated form
A preferred example is
Formula T8E:
[Ma]Gal(31-3/4[Na]nG1cNAcAxHex(NAc)n
wherein
wherein m, n and p are integers 0, or 1, independently
Hex is Gal or Glc,
X is linkage position
M and N are monosaccharide residues being
independently nothing (free hydroxyl groups at the positions)
and/or
SA which is Sialic acid linked to 3-position of Gal or/and 6-position of
HexNAc
and/or
Fuc (L-fucose) residue linked to 2-position of Gal
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and/or 3 or 4 position of G1cNAc, when Gal is linked to the other position (4
or 3),
and HexNAc is G1cNAc, or 3-position of Glc when Gal is linked to the other
position (3),
with the provision that sum of m and n is 2
preferably m and n are 0 or 1, independently.
A is anomeric structure alfa or beta,X is linkage position 2, 3,or 6
And Hex is hexopyranosyl residue Gal, or Man, and n is integer being 0 or 1,
with the provisions
that when n is 1 then AxHexNAc is (36GaLNAc, when Hex is Man, then AxHex is
(32Man, and
when Hex is Gal, then AxHex is 03Ga1 or 06Gal.
The most preferred structures are according to the formula
Formula T8Ebeta, wherein the anomeric structure is beta:
[Ma]Gal(31-3/4[Na]nG1cNAc(3xHex(NAc)n
A preferred group of type II Lactosmines are (32-linked on Man or N-glycans or
(36-linked on
Gal(NAc) in O-glycan/poly-LacNac structures according to the
Formula T10E
[Ma]Gal(31-4[Na]nG1cNAcAxHex(NAc)n
Formula T10EMan:
[Ma]mGal(31-4[Na]nG1cNAc(32Man
and
Formula T10EGa1(NAc):
[Ma]mGal(31-4[Na]nG1cNAc(36Ga1(NAc)
and further elongated structures according to the invention.
A preferred group of type I Lactosmines are (33- on Gal
According to the Formula T9E
[Ma]Gal(31-3[Na]nG1cNAc(33Gal
The preferred subgroups of the elongation structures includes i) similar
structural epitopes present
on O-glycans, polylactosamine and glycolipid cores: (33/6Gal or 06Ga1NAc; with
preferred further
subgroups ia) (36GalNAc/(36Gal and ib) (33Ga1; ii) N-glycan type epitope
(32Man; and iii)
globoseries epitopes a3Gal or a4Ga1. The groups are preferred for structural
similarity on possible
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36
cross reactivity within the groups, which can be used for increasing labeling
intensity when
background materials are controlled to be devoid of the elongated structure
types.
Useful binder specifities including lectin and elongated antibody epitopes is
available from reviews
and monographs such as (Debaray and Montreuil (1991) Adv. Lectin Res 4, 51-96;
"The molecular
immunology of complex carbohydrates" Adv Exp Med Biol (2001) 491 (ed Albert M
Wu) Kluwer
Academic/Plenum publishers, New York; "Lectins" second Edition (2003) (eds
Sharon, Nathan and
Lis, Halina) Kluwer Academic publishers Dordrecht, The Netherlands and
internet databases such
as pubmed/espacenet or antibody databases such as wvvw.gl-Tco.is.ritsumer.ac.i
/e ito7e/, which list
monoclonal antibody glycan specificities).
Combination of the preferred elongated
The invention is directed in apreferred embodiment combined use of the
preferred structures and
elongated structures for recognition of stem cells. In a preferred embodiment
at least one type I
LacNAc or type II lacNAc structure are used, in another preferred embodiment a
non-reducing end
non-modified LacNAc is used with a2Fucosylated LacNAc, Lewis x or sialylated
LacNAc, in a
preferred embodiment a2Fucosylated type I and type 11 LacNAc are used. The
inventors used factor
analysis to produce more preferred combinations according to the invention
including use of
complex type glycans together with high mannose or Low mannose glycan. In a
preferred
embodiment a LacNAc structure is used togerher with a preferred glycolipid
structure, preferably
globotriose type. The invention is preferably directed to recognition of
differentiation and/or cell
culture condition assosiceted changes in the stem cells.
Preferred elon . ag ted epitopes
It is realized that elongated glycan epitopes are useful for recognition of
the embryonic type stem
cells according to the invention. The invention is directed to the use of -
some of the structures for
characterizing all the cell types, while certain structural motifs are more
common at a specific
differentiation stage.
It is further realized that some of the terminal structures are expressed at
especially high levels and
thus especially useful for the recognition of one or several types of cells.
The terminal epitopes and the glycan types are listed in Table 21, based on
the structural analysis of
the glycan types following preferred elongated structural epitopes that are
preferred as novel
markers for embryonal type stem cells and for the uses according to the
invention.
Preferred terminal Gal(33/4 Structures
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37
Type II N-acetyllactosamine based structures
Terminal type II N-acetyllactosamine structures
The invention revealed preferred type II N-acetyllactosamines including
specific 0-glycan, N-
glycan and glycolipid epitopes. The invention is in a preferred embodiment
especially directed to
abundant 0-glycan and N-glycan epitopes. The invention is further directed to
the recognition of a
characteristic glycolipid type II LacNAc terminal. The invention is especially
directed to the use of
the Type II LacNAc for recognition of non-differentiated embryonal type stem
cells (stage I) and
similar cells or for the analysis of the differentiation stage. It is however
realized that substantial
amounts of the structures are present in the more differentiated cells as
well.
Elongated type II LacNAc structures are especially expressed on N-glycans.
Preferred type II
LacNAc structures are 02-linked to the biantennary N-glycan core structure,
including the preferred
epitopes Gal(34GlcNAc(32Man, Gal(34GlcNAc(32Mana, Gal(34GlcNAc(32Mana3/6Man
and
Gal(34GlcNAc(32Mana3/6Man(34
The invention further revealed novel 0-glycan epitopes with terminal type II N-
acetyllactosamine
structures expressed effectively on the embryonal type cells. The analysis of
the 0-glycan
structures revealed especially core II N-acetyllactosamines with the terminal
structure. The
preferred elongated type II N-acetyllactosamines thus includes
Gal(34G1cNAc(36GaLNAc,
Gal(34G1cNAc(36Ga1NAca, Gal(34G1cNAc(36(Gal(33)Ga1NAc, and
Gal04G1cNAc(36(Gal(33)GalNAca.
The invention further revealed the presence of type II LacNAc on glycolipids.
The present
invention reveals for the first time terminal type II N-acetyllactosamine on
glycolipids of stem cells.
The neolacto glycolipid family is an important glycolipid family
characteristically expressed on
certain tissues but not on others.
The preferred glycolipid structures include epitopes, preferably non-reducing
end terminal epitopes
of linear neolactotetraosyl ceramide and elongated variants thereof
Gal(34G1cNAc(33Gal,
Gal(34GlcNAc(33Ga1(34, Gal(34G1cNAc(33Gal(34Glc(NAc),
Gal(34G1cNAc(33Ga1(34Glc, and
Gal(34G1cNAc(33Ga1(34G1cNAc. It is furher realized that specific reagents
recognizing the linear
polylactosamines can be used for the recognition of the structures, when these
are linked to protein
linked glycans. In a preferred embodiment the invention is directed to the
poly-N-
acetyllactosamines linked to N-glycans, preferably (32-linked structures such
as
Gal(34GlcNAc(33Ga1(34G1cNAc(32Man on N-glycans. The invention is further
directed to the
characterization of the poly-N-acetyllactosamine structures of the preferred
cells and their
modification by SAa3, SAa6, Fuca2 to non-reducing end Gal and by Fuca3 to
G1cNAc residues.
The invention is preferably directed to recognition of tetrasaccharides,
hexasaccharides, and
octasaccharides. The invention further revealed branched glycolipid
polylactosamines including
terminal type II LacNAc epitopes, preferably these include Gal(34G1cNAc(36Ga1,
Gal04GlcNAc(36Ga1(3, Gal(34G1cNAc(36(Gal(34G1cNAc(33)Gal, and
Gal(34G1cNAc(36(Gal(34GlcNAc(33)Gal(33,
Gal(34G1cNAc(36(Gal(34G1cNAc(33)Gal(34G1c(NAc),
Gal(34G1cNAc(36(Gal(34GlcNAc(33)Gal(34G1c, and
Gal(34GlcNAc(36(Gal(34GlcNAc(33)Gal(34G1cNAc.
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38
It is realized that antibodies specifically binding to the linear or branched
poly-N-
acetyllactosamines are well known in the art. The invention is further
directed to reagents
recognizing both branched polyLacNAcs and core II 0-glycans with similar
(36Ga1(NAc) epitopes.
Lewis x structures
Elongated Lewis x structures are especially expressed on N-glycans. Preferred
Lewis x structures
are 02-linked to the biantennary N-glycan core structure, including the
preferred structures
Gal(34(Fuca3)G1cNAc(32Man,
Gal(34(Fuca3)G1cNAc(32Mana, Gal(34(Fuca3)G1cNAc(32Mana3/6Man,
Gal(34(Fuca3)G1cNAc(32Mana3/6Man(34
The invention further revealed the presence of Lewis x on glycolipids. The
preferred glycolipid
structures include Gal(Fuca3)(34G1cNAc(33Gal, Gal(34(Fuca3)G1cNAc(33Ga1,
Gal04(Fuca3)G1cNAc(33Ga1(34, Gal(34(Fuca3)G1cNAc(33Ga1(34G1c(NAc),
Gal(34(Fuca3)G1cNAc(33Ga1(34GIc, and Gal(34(Fuca3)G1cNAc(33Ga1(34G1cNAc.
The invention further revealed the presence of Lewis x on 0-glycans. The
preferred 0-glycan
structures include preferably the core II structures
Gal(34(Fuca3)G1cNAc(36GaLNAc,
Gal(34(Fuca3)G1cNAc(36Ga1NAca, Gal(34(Fuca3)G1cNAc(36(Gal(33)Ga1NAc, and
Gal(34(Fuca3)G1cNAc(36(Gal(33)Ga1NAca.
H type II structures
Specific elongated H type II structural epitopes are especially expressed on N-
glycans. Preferred H
type II structures are 02-linked to the biantennary N-glycan core structure,
Fuca2Ga1(34G1cNAc(32Mana3/6Manp4
The invention further revealed the presence of H type II on glycolipids. The
preferred glycolipid
structures includes Fuca2Gal(34G1cNAc(33Gal, Fuca2Ga1(34G1cNAc(33Ga1,
Fuca2Ga1(34G1cNAc(33Ga1(34, Fuca2Gal(34G1cNAc(33Gal(34G1c(NAc),
Fuca2Ga1(34G1cNAc(33Ga1(34Glc, and Fuca2Ga1(34GlcNAc(33Gal(34G1cNAc.
The invention further revealed the presence of H type II on 0-glycans. The
preferred 0-glycan
structures include preferably core II structures Fuca2Ga1(34G1cNAc(36Ga1NAc,
Fuca2Ga1(34G1cNAc(36Ga1NAca, Fuca2Ga1(34G1cNAc(36(Gal(33)Ga1NAc, and
Fuca2Ga1(34G1cNAc(36(Gal(33)Ga1NAca.
Sialylated type II N-acetyllactosamine structures
The invention revealed preferred sialylated type II N-acetyllactosamines
including specific 0-
glycan, N-glycan and glycolipid epitopes. The invention is in a preferred
embodiment especially
directed to abundant 0-glycan and N-glycan epitopes. SA refers here to sialic
acid, preferably
Neu5Ac or Neu5Gc, more preferably Neu5Ac. The sialic acid residues are SAa3Ga1
or SAa6Ga1,
it is realized that these structures when presented as specific elongated
epitopes form characteristic
terminal structures on glycans.
Sialylated type II LacNAc structural epitopes are especially expressed on N-
glycans. Preferred type
II LacNAc structures are (32-linked to biantennary N-glycan core structure,
including the preferred
terminal epitopes SAa3/6Gal(34G1cNAc(32Man, SAa3/6Gal(34G1cNAc(32Mana, and
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39
SAa3/6Gal(34GlcNAc(32Mana3/6Man(34. The invention is directed to both SAa3-
structures
(SAa3Gal(34GlcNAc(32Man, SAa3Gal(34GlcNAc(32Mana, and
SAa3Gal(34GlcNAc(32Mana3/6Man(34) and SAa6-epitopes
(SAa6Gal(34GIcNAc(32Man, SAa6Gal(34GIcNAc(32Mana, and
SAa6Gal(34GlcNAc(32Mana3/6Man(34) on N-glycans.
The SAa3-N-glycan epitopes are preferred for the analysis of the non-
differentiated stage I
embryonic type cells. The SAa6-N-glycan epitopes are preferred for analysis of
the
differentiated/or differentiating embryonic type cells, such as embryoid
bodies and stage III
differentiated embryonic type cells. It is realized that the combined analysis
of both types of N-
glycans is useful for the characterization of the embryonic type stem cells.
The invention further revealed novel 0-glycan epitopes with terminal
sialylated type II N-
acetyllactosamine structures expressed effectively on the embryonal type
cells. The analysis of 0-
glycan structures revealed especially core II N-acetyllactosamines with the
terminal structure. The
preferred elongated type II sialylated N-acetyllactosamines thus include
SAa3/6Gal(34GlcNAc(36Ga1NAc, SAa3/6Gal(34G1cNAc(36GaLNAca,
SAa3/6Ga1(34GlcNAc(36(Gal(33)GaINAc, and SAa3/6Ga1(34GlcNAc(36(Ga1(33)GalNAca.
The
SAa3-structures were revealed as preferred structures in context of the 0-
glycans including
SAa3Gal(34GlcNAc(36GalNAc, SAa3Gal(34GlcNAc(36GalNAca,
SAa3Gal(34GlcNAc(36(Gal(33)GaLNAc, and SAa3Gal(34G1cNAc(36(Gal(33)GaLNAca.
Specific preferred tetrasaccharide type II lactosamine epitopes
It is realized that highly effective reagents can in a preferred embodiment
recognize epitopes which
are larger than a trisaccharide. Therefore the invention is further directed
to the branched terminal
type II lactosamine derivatives Lewis y Fuca2Ga1(34(Fuca3)G1cNAc and sialyl-
Lewis x
SAa3Ga1(34(Fuca3)G1cNAc as preferred elongated or large glycan structural
epitopes. It is realized
that the structures are combinations of preferred termina trisaccharide sialyl-
lactosamine, H-type II
and Lewis x epitopes. The analysis of the epitopes is preferred as
additionally useful method in the
context of analysis of other terminal type II epitopes. The invention is
especially directed to -further
defining the core structures carrying the Lewis y and sialyl-Lewis x epitopes
on various types of
glycans and optimizing the recognition of the structures by including the
recognition of the
preferred glycan core structures.
Structures analogous to the type II lactosamines
The invention is further directed to the recognition of elongated epitopes
analogous to the type II N-
acetyllactosamines including LacdiNAc especially on N-glycans and
lactosylceramide
(Gal(34Glc(3Cer) glycolipid structure. These share similarity with LacNAc the
only difference being
the number of NAc residues on the monosaccharide residues.
LacdiNAc structures
It is realized that LacdiNac is relatively rare and characteristic glycan
structure and it is therefore
especially preferred for the characterization of the embryonic type cells. The
invention revealed the
presence of LacdiNAc on N-glycans at least as 02-linked terminal epitope. The
structures were
characterized by specific glycosidase cleavages. The LacdiNAc structures have
same mass as
structures with two terminal G1cNAc containing structures in structural Table
13, Table 13 includes
representative structures indicating only single isomeric structures for a
specific mass number. The
preferred elongated LacdiNAc epitopes thus include-S GaLNAc(34G1cNAc(32Man,
GaINAc(34GlcNAc(32Mana, and Ga1NAc(34G1cNAc(32Mana3/6Man(34. The invention
further
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revealed fucosylation of LacdiNAc containing glycan structures and the
preferred epitopes thus
further include Ga1NAc(34(Fuca3)G1cNAc(32Man, Ga1NAc(34(Fuca3)G1cNAc(32Mana,
Ga1NAc(34(Fuc(x3)GIcNAc(32Mana3/6Man(34
Ga1NAc(Fuca3)(34G1cNAc(32Mana3/6Man(34. It is
realized that presence of (36-linked sialic acid of LacNac of structure with
mass number 2263, table
13 indicates that at least part of the fucose is present on the LacdiNAc arm
of the molecule based on
the competing nature of a6-sialylation and a3-fucosylation on enzyme
specificity level (alternative
assignment presented in the Table 13).
Type I N-acetyllactosamine based structures
Terminal type I N-acetyllactosamine structures
The invention revealed preferred type I N-acetyllactosamines including
specific 0-glycan, N-glycan
and glycolipid epitopes. The invention is in a preferred embodiment especially
directed to abundant
glycolipid epitopes. The invention is further preferably directed to the
recognition of characteristic
0-glycan type I LacNAc terminals.
The invention is especially directed to the use of the Type I LacNAc for the
recognition of non-
differentiated embryonal type stem cells (stage I) and similar cells or for
the analysis of the
differentiation stage. It is however realized that substantial amount of the
structures are present in
the more differentiated cells as well.
The invention further revealed novel 0-glycan epitopes with terminal type I N-
acetyllactosamine
structures expressed effectively on the embryonal type cells. The analysis of
0-glycan structures
revealed especially core II N-acetyllactosamines with the terminal structure
on type II lactosamine.
The preferred elongated type I N-acetyllactosamines thus includes
Gal(33GlcNAc(33Ga1(34G1cNAc(36Ga1NAc, Gal(33GlcNAc(33Ga1(34G1cNAc(36Ga1NAca,
Gal(33GlcNAc(33Ga1GIcNAc(36(Gal(33)Ga1NAc, and
Gal(33GlcNAc(33Ga1(34G1cNAc(36(Gal(33)Ga1NAca.
The invention further revealed the presence of type I LacNAc on glycolipids.
The present invention
reveals for the first time terminal type I N-acetyllactosamine on glycolipids.
The Lacto glycolipid
family is an important glycolipid family characteristically expressed on
certain tissue but not on
others.
The preferred glycolipid structures include-epitopes, preferably non-reducing
end terminal epitopes,
of linear lactoteraosyl ceramide and elongated variants thereof
Gal(33G1cNAc(33Ga1,
Gal(33GlcNAc(33Ga1(34, Ga1(33G1cNAc(33Ga1(34Glc(NAc),
Gal(33G1cNAc(33Ga1(34G1c, and
Gal(33GlcNAc(33Ga1(34G1cNAc. It is further realized that specific reagents
recognizing the linear
polylactosamines can be used for the recognition of the structures, when these
are linked to protein
linked glycans. It is especially realized that the terminal tri-and
tetrasaccharide epitopes on the
preferred 0-glycans and glycolipids are essentially the same. The invention is
in a preferred
embodiment directed to the recognition of the both structures by the same
binding reagent such as a
monoclonal antibody
The invention is further directed to the characterization of the terminal type
I poly-N-
acetyllactosamine structures of the preferred cells and their modification by
SAa3, Fuca2 to non-
reducing end Gal and by SAa6 or Fuca3 to G1cNAc residues and other core glycan
structures of
the derivatized type I N-acetyllactosamines.
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41
A preferred elongated type I LacNAc structure is expressed on N-glycans.
Preferred type I LacNAc
structures are (32-linked to the biantennary N-glycan core structure, the
preferred epitopes being
Gal(33GlcNAc(32Man, Gal(33G1cNAc(32Mana and Gal(33G1cNAc(32Mana3/6Man(34.
Fucosylated type I LacNAcs
Lewis a structures
The invention revealed the presence of Lewis a structures on glycolipids. The
invention is further
directed to related poly-N-acetyllactosamine structures with similar terminal
epitopes. The preferred
glycolipid structures includes Ga1(33(Fuca4)(3G1cNAc(33Gal,
Ga1(33(Fuc(x4)(3G1cNAc(33Ga1,
Gal(33(Fuca4)(3G1cNAc(33Ga1(34, Gal(33(Fuca4)(3GlcNAc(33Gal(34G1c(NAc),
Gal(33(Fuca4)(3G1cNAc(33Ga1(34G1c, and Gal(33(Fuca4)(3G1cNAc(33Gal(34GlcNAc.
The invention is further directed to the presence of Lewis a on elongated 0-
glycans. The preferred
0-glycan polylactosamine type structures include preferably the core 11
structures
Gal(33(Fuca4)G1cNAc(33Ga1(34G1cNAc(36Ga1NAc,
Gal(33(Fuca4)G1cNAc(33Ga1(34G1cNAc(36Ga1NAca,
Gal(33(Fuca4)G1cNAc(33Ga1(34G1cNAc(36(Gal(33)Ga1NAc, and
Gal(33(Fuca4)G1cNAc(33Ga1(34G1cNAc(36(Gal(33)Ga1NAca.
H type I structures
A Preferred elongated H type I structure is on lacto series glycolipids or
related poly-N-
acetyllactosamine structures. The preferred glycolipid/polylactosamine
structures include-s
Fuca2Ga1(33G1cNAc(33Ga1, Fuca2Ga1(33G1cNAc(33Ga1, Fuca2Gal(33GlcNAc(33Gal(34,
Fuca2Ga1(33G1cNAc(33Ga1(34G1c(NAc), Fuca2Gal(33G1cNAc(33Gal(34Glc, and
Fuca2Ga1(33 G1cNAc(33 Ga1(34G1cNAc.
The invention is further directed to the presence of H type I on elongated 0-
glycans. The preferred
0-glycan polylactosamine type structures include preferably the core 11
structures
Fuca2Ga1(33G1cNAc(33Ga1(34G1cNAc(36GalNAc,
Fuca2Ga1(33G1cNAc(33Ga1(34G1cNAc(36GalNAca,
Fuca2Ga1(33G1cNAc(33Ga1(34G1cNAc(36(Gal(33)Ga1NAc, and
Fuca2Ga1(33G1cNAc(33Ga1(34G1cNAc(36(Gal(33)GalNAca.
Specific preferred tetrasaccharide type I lactosamine epitopes
It is realized that highly effective reagents can in a preferred embodiment
recognize epitopes which
are larger than a trisaccharide. Therefore the invention is further directed
to the branched terminal
type I lactosamine derivatives Lewis b Fuca2Ga1(33(Fuca4)G1cNAc and sialyl-
Lewis a
SAa3Ga1(33(Fuca4)G1cNAc as preferred elongated or large glycan structural
epitopes. It realized
that the structures are combinations of preferred terminal trisaccharide
sialyl-lactosamine, H-type I
and Lewis a epitopes. The analysis of the epitopes is preferred as
additionally useful method in the
context of analysis of other terminal type I epitopes. The invention is
especially directed to-further
defining the core structures carrying the type Lewis b and sialyl-Lewis a
epitopes on various types
of glycans and optimizing the recognition of the structures by including the
recognition of preferred
glycan core structures. The invention revealed that at least some of the
sialyl-Lewis a epitopes are
scarce on stage I cells and the structure is associated more with
differentiated cell types.
As used herein, "binder", "binding agent" and "marker" are used
interchangeably.
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42
Antibodies
Various procedures known in the art may be used for the production of
polyclonal antibodies to
peptide motifs and regions or fragments thereof. For the production of
antibodies, any suitable host
animal (including but not limited to rabbits, mice, rats, or hamsters) are
immunized by injection
with a peptide (immunogenic fragment). Various adjuvants may be used to
increase the
immunological response, depending on the host species, including but not
limited to Freund's
(complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide,
surface active
substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions,
keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG
{Bacille
Calmette-Guerin) and Corynebacterium parvum.
A monoclonal antibody to a peptide or glycan motif(s) may be prepared by using
any technique
which provides for the production of antibody molecules by continuous cell
lines in culture. These
include but are not limited to the hybridoma technique originally described by
K6hler et al.,
(Nature, 256: 495-497, 1975), and the more recent human B-cell hybridoma
technique (Kosbor et
al., Immunology Today, 4: 72, 1983) and the EBV-hybridoma technique (Cole et
al., Monoclonal
Antibodies and Cancer Therapy, Alan R Liss, Inc., pp. 77-96, 1985), all
specifically incorporated
herein by reference. Antibodies also may be produced in bacteria from cloned
immunoglobulin
cDNAs. With the use of the recombinant phage antibody system it may be
possible to quickly
produce and select antibodies in bacterial cultures and to genetically
manipulate their structure.
When the hybridoma technique is employed, myeloma cell lines may be used. Such
cell lines suited
for use in hybridoma-producing fusion procedures preferably are non-antibody-
producing, have
high fusion efficiency, and exhibit enzyme deficiencies that render them
incapable of growing in
certain selective media which support the growth of only the desired fused
cells (hybridomas). For
example, where the immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-
Ag8.653,
NSl/l.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-I 1, MPC11-X45-GTG 1.7 and S194/5XXO
Bul; for
rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-
GRG2,
LICR-LON-HMy2 and UC729-6 all may be useful in connection with cell fusions.
In addition to the production of monoclonal antibodies, techniques developed
for the production of
"chimeric antibodies", the splicing of mouse antibody genes to human antibody
genes to obtain a
molecule with appropriate antigen specificity and biological activity, can be
used (Morrison et al,
Proc Natl Acad Sd 81 : 6851-6855, 1984; Neuberger et al, Nature 312: 604-608,
1984; Takeda et al,
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43
Nature 314: 452-454; 1985). Alternatively, techniques described for the
production of single- chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce influenza-
specific single chain
antibodies.
Antibody fragments that contain the idiotype of the molecule may be generated
by known
techniques. For example, such fragments include, but are not limited to, the
F(ab')2 fragment which
may be produced by pepsin digestion of the antibody molecule; the Fab'
fragments which may be
generated by reducing the disulfide bridges of the F(ab')2 fragment, and the
two Fab fragments
which may be generated by treating the antibody molecule with papain and a
reducing agent.
Non-human antibodies may be humanized by any methods known in the art. A
preferred
"humanized antibody" has a human constant region, while the variable region,
or at least a
complementarity determining region (CDR), of the antibody is derived from a
non-human species.
The human light chain constant region may be from either a kappa or lambda
light chain, while the
human heavy chain constant region may be from either an IgM, an IgG (IgGl,
IgG2, IgG3, or IgG4)
an IgD, an IgA, or an IgE immunoglobulin.
Methods for humanizing non-human antibodies are well known in the art (see
U.S. PatentNos.
5,585,089, and 5,693,762). Generally, a humanized antibody has one or more
amino acid residues
introduced into its framework region from a source which is non-human.
Humanization can be
performed, for example, using methods described in Jones et al. {Nature 321:
522-525, 1986),
Riechmann et al, {Nature, 332: 323-327, 1988) and Verhoeyen et al. Science
239:1534-1536,
1988), by substituting at least a portion of a rodent complementarity-
determining region (CDRs) for
the corresponding regions of a human antibody. Numerous techniques for
preparing engineered
antibodies are described, e.g. , in Owens and Young, J. Immunol. Meth.,
168:149-165, 1994.
Further changes can then be introduced into the antibody framework to modulate
affinity or
immunogenicity.
Likewise, using techniques known in the art to isolate CDRs, compositions
comprising CDRs are
generated. Complementarity determining regions are characterized by six
polypeptide loops, three
loops for each of the heavy or light chain variable regions. The amino acid
position in a CDR and
framework region is set out by Kabat et al., "Sequences of Proteins of
Immunological Interest,"
U.S. Department of Health and Human Services, (1983), which is incorporated
herein by reference.
For example, hypervariable regions of human antibodies are roughly defined to
be found at residues
28 to 35, from residues 49-59 and from residues 92-103 of the heavy and light
chain variable
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44
regions (Janeway and Travers, Immunobiology, 2nd Edition, Garland Publishing,
New York, 1996).
The CDR regions in any given antibody may be found within several amino acids
of these
approximated residues set forth above. An immunoglobulin variable region also
consists of
"framework" regions surrounding the CDRs. The sequences of the framework
regions of different
light or heavy chains are highly conserved within a species, and are also
conserved between human
and murine sequences.
Compositions comprising one, two, and/or three CDRs of a heavy chain variable
region or a light
chain variable region of a monoclonal antibody are generated. Polypeptide
compositions comprising
one, two, three, four, five and/or six complementarity determining regions of
a monoclonal
antibody secreted by a hybridoma are also contemplated. Using the conserved
framework sequences
surrounding the CDRs, PCR primers complementary to these consensus sequences
are generated to
amplify a CDR sequence located between the primer regions. Techniques for
cloning and
expressing nucleotide and polypeptide sequences are well-established in the
art [see e.g., Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor, New York
(1989)]. The amplified CDR sequences are ligated into an appropriate plasmid.
The plasmid
comprising one, two, three, four, five and/or six cloned CDRs optionally
contains additional
polypeptide encoding regions linked to the CDR.
Preferably, the antibody is any antibody specific for a glycan structure of
Formula (I) or a fragment
thereof. The antibody used in the present invention encompasses any antibody
or fragment thereof,
either native or recombinant, synthetic or naturally-derived, monoclonal or
polyclonal which retains
sufficient specificity to bind specifically to the glycan structure according
to Formula (I) which is
indicative of stem cells. As used herein, the terms "antibody" or "antibodies"
include the entire
antibody and antibody fragments containing functional portions thereo The
term "antibody"
includes any monospecific or bispecific compound comprised of a sufficient
portion of the light
chain variable region and/or the heavy chain variable region to effect binding
to the epitope to
which the whole antibody has binding specificity. The fragments can include
the variable region of
at least one heavy or light chain immunoglobulin polypeptide, and include, but
are not limited to,
Fab fragments, F(ab')2 fragments, and Fv fragments.
The antibodies can be conjugated to other suitable molecules and compounds
including, but not
limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens,
fluorochromes, metal
compounds, radioactive compounds, chromatography resins, solid supports or
drugs. The enzymes
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that can be conjugated to the antibodies include, but are not limited to,
alkaline phosphatase,
peroxidase, urease and .beta.-galactosidase. The fluorochromes that can be
conjugated to the
antibodies include, but are not limited to, fluorescein isothiocyanate,
tetramethylrhodamine
isothiocyanate, phycoerythrin, allophycocyanins and Texas Red. For additional
fluorochromes that
can be conjugated to antibodies see Haugland, R. P. Molecular Probes: Handbook
of Fluorescent
Probes and Research Chemicals (1992-1994). The metal compounds that can be
conjugated to the
antibodies include, but are not limited to, ferritin, colloidal gold, and
particularly, colloidal
superparamagnetic beads. The haptens that can be conjugated to the antibodies
include, but are not
limited to, biotin, digoxigenin, oxazalone, and nitrophenol. The radioactive
compounds that can be
conjugated or incorporated into the antibodies are known to the art, and
include but are not limited
to technetium 99m, 125 I and amino acids comprising any radionuclides,
including, but not
limited to l4 C, 3 H and35 S.
Antibodies to glycan structure(s) of Formula (I) may be obtained from any
source. They may be
commercially available. Effectively, any means which detects the presence of
glycan structure(s) on
the stem cells is with the scope of the present invention. An example of such
an antibody is a H type
1(clone 17-206; GF 287) antibody from Abcam.
Preferred N-glycan structure types
The invention revealed N-glycans with common core structure of N-glycans,
which change
according to differentiation and/or individual specific differences.
The N-glycans of embryonic stem cells comprise core structure comprising
Man(34GlcNAc structure in the core structure of N-linked glycan according to
the
Formula CGN :
[Mana3]ni(Mana6) nzMan(34G1cNAc(34(Fuca6)n3G1cNAcxR,
wherein nl, n2 and n3 are integers 0 or 1, independently indicating the
presence or
absence of the residues, and
wherein the non-reducing end terminal Mana3/Mana6- residues can be elongated
to the
complex type, especially biantennary structures or to mannose type (high-Man
and/or low Man)
or to hybrid type structures (for the analysis of the status of stem cells
and/or manipulation of
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46
the stem cells), wherein xR indicates reducing end structure of N-glycan
linked to protein or
peptide such as (3Asn or (3Asn-peptide or (3Asn-protein, or free reducing end
of N-glycan or
chemical derivative of the reducing end produced for analysis.
The preferred Mannose type glycans are according to the formula:
Formula M2:
[Ma2]õ, [Ma3 ],,z{ [Ma2]n3 [Ma6)]n4} [Ma6]ns {[Ma2]n6 [Ma2]õ7[Ma3 ]ng}
M(34GN(34 [ {Fuca6} ]mGNyRz
wherein nl, n2, n3, n4, n5, n6, n7, n8, and m are either independently 0 or 1;
with the provision that
when n2 is 0, also nl is 0; when n4 is 0, also n3 is 0; when n5 is 0, also nl,
n2, n3, and n4 are 0;
when n7 is 0, also n6 is 0; when n8 is 0, also n6 and n7 are 0;
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-glycoside
derivative such as asparagine N-glycosides including asparagines N-glycoside
amino acid and/or
peptides derived from protein;
[] indicates determinant either being present or absent depending on the value
of nl, n2, n3, n4, n5,
n6, n7, n8, and m; and
{} indicates a branch in the structure;
M is D-Man, GN is N-acetyl-D-glucosamine and Fuc is L-Fucose,
and the structure is optionally a high mannose structure, which is further
substituted by glucose
residue or residues linked to mannose residue indicated by n6.
Several preferred low Man glycans described above can be presented in a single
Formula:
[Ma3]nz{[Ma6)]n4}[Ma6]ns{[Ma3]ng}M(34GN(34[{Fuca6}]mGNyRz
wherein n2, n4, n5, n8, and m are either independently 0 or 1; with the
provision that when n5 is 0,
also n2, and n4 are 0;the sum of n2, n4, n5, and n8 is less than or equal to
(m + 3); [] indicates
determinant either being present or absent depending on the value of n2, n4,
n5, n8, and m; and
{} indicates a branch in the structure;
y and R2 are as indicated above.
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Preferred non-fucosylated low-mannose glycans are according to the formula:
[Ma3]nz([Ma6)]n4)[Ma6]n5{[Ma3]ng}M(34GN(34GNyR2
wherein n2, n4, n5, n8, and m are either independently 0 or 1,
with the provision that when n5 is 0, also n2 and n4 are 0, and preferably
either n2 or n4 is 0,
[] indicates determinant either being present or absent
depending on the value of , n2, n4, n5, n8,
{} and Q indicates a branch in the structure,
y and R2 are as indicated above.
Preferred individual structures of non-fucosylated low-mannose glycans
Special small structures
Small non-fucosylated low-mannose structures are especially unusual among
known N-linked
glycans and characteristic glycan group useful for separation of cells
according to the present
invention. These include:
M(34GN(34GNyR2
Ma6M(34GN(34GNyR2
Ma3M(34GN(34GNyR2 and
Ma6{Ma3}M(34GN(34GNyR2.
M(34GN(34GNyR2 trisaccharide epitope is a preferred common structure alone and
together with its mono-
mannose derivatives Ma6M(34GN(34GNyR2 and/or Ma3M(34GN(34GNyR2, because these
are
characteristic structures commonly present in glycomes according to the
invention. The invention is
specifically directed to the glycomes comprising one or several of the small
non-fucosylated low-mannose
structures. The tetrasaccharides are in a specific embodiment preferred for
specific recognition directed to a-
linked, preferably 0/6-linked Mannoses as preferred terminal recognition
element.
Special laY,ee structures
The invention further revealed large non-fucosylated low-mannose structures
that are unusual
among known N-linked glycans and have special characteristic expression
features among the
preferred cells according to the invention. The preferred large structures
include
[Ma3]n2([Ma6]n4)Ma6{Ma3}M(34GN(34GNyRz
more specifically
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Ma6Ma6{Ma3}M(34GN(34GNyR2
Ma3Ma6{Ma3}M(34GN(34GNyR2 and
Ma3(Ma6)Ma6{Ma3}M(34GN(34GNyR2.
The hexasaccharide epitopes are preferred in a specific embodiment as rare and
characteristic structures in
preferred cell types and as structures with preferred terminal epitopes. The
heptasaccharide is also preferred
as a structure comprising a preferred unusual terminal epitope Ma3(M(x6)Ma
useful for analysis of cells
according to the invention.
Preferred fucosylated low-mannose glycans are derived according to the
formula:
[Ma3]n2{[Ma6]n4} [Ma6]ns{[Ma3]ng}M(34GN(34(Fuc(x6)GNyRz
wherein n2, n4, n5, n8, and m are either independently 0 or l,with the
provision that when n5 is 0,
also n2 and n4 are 0,
[] indicates determinant either being present or absent
depending on the value of n2, n4, n5, n8, and m;
{} and ( indicate a branch in the structure.
Preferred individual structures of fucosvlated low-mannose kl cy ans
Small fucosylated low-mannose structures are especially unusual among known N-
linked glycans
and form a characteristic glycan group useful for separation of cells
according to the present
invention. These include:
M(34GN(34(Fuc(x6)GNyR2
Ma6M(34GN(34(Fuca6)GNyR2
Ma3M(34GN(34(Fuc(x6)GNyR2 and
Ma6{Ma3}M(34GN(34(Fuca6)GNyR2.
M(34GN(34(Fuc(x6)GNyR2 tetrasaccharide epitope is a preferred common structure
alone and together with
its monomannose derivatives Ma6M(34GN(34(Fuca6)GNyR2 and/or
Ma3M(34GN(34(Fuca6)GNyR2,
because these are commonly present characteristic structures in glycomes
according to the invention. The
invention is specifically directed to the glycomes comprising one or several
of the small fucosylated low-
mannose structures. The tetrasaccharides are in a specific embodiment
preferred for specific recognition
directed to a-linked, preferably a3/6-linked Mannoses as preferred terminal
recognition element.
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Special large structures
The invention further revealed large fucosylated low-mannose structures that
are unusual among
known N-linked glycans and have special characteristic expression features
among the preferred
cells according to the invention. The preferred large structures include
[Ma3]n2([Ma6]n4)Ma6{Ma3}M(34GN(34(Fuca6)GNyR2
more specifically
Ma6Ma6{Ma3}M(34GN(34(Fuca6)GNyR2
Ma3Ma6{Ma3}M(34GN(34(Fuc(x6)GNyR2 and
Ma3(Ma6)Ma6{Ma3{M(34GN(34(Fuca6)GNyR2.
The heptasaccharide epitopes are preferred in a specific embodiment as rare
and characteristic structures in
preferred cell types and as structures with preferred terminal epitopes. The
octasaccharide is also preferred as
structure comprising a preferred unusual terminal epitope Ma3(M(x6)Ma useful
for analysis of cells
according to the invention.
Preferred non-reducing end terminal Mannose-epitopes
The inventors revealed that mannose-structures can be labeled and/or otherwise
specifically
recognized on cell surfaces or cell derived fractions/materials of specific
cell types. The present
invention is directed to the recognition of specific mannose epitopes on cell
surfaces by reagents
binding to specific mannose structures on cell surfaces.
The preferred reagents for recognition of any structures according to the
invention include specific
antibodies and other carbohydrate recognizing binding molecules. It is known
that antibodies can be
produced for the specific structures by various immunization and/or library
technologies such as
phage display methods representing variable domains of antibodies. Similarly
with antibody library
technologies, including aptamer technologies and including phage display for
peptides, exist for
synthesis of library molecules such as polyamide molecules including peptides,
especially cyclic
peptides, or nucleotide type molecules such as aptamer molecules.
The invention is specifically directed to specific recognition of high-mannose
and low-mannose
structures according to the invention. The invention is specifically directed
to recognition of non-
reducing end terminal Mana-epitopes, preferably at least disaccharide
epitopes, according to the
formula:
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[Ma2]m, [Max]m2[Ma6]m3 {{[Ma2]m9[Ma2]m8[Ma3]m7}mjo(M(34[GN]m4)m5}m6yR2
wherein ml, m 2, m3, m4, m5, m6, m7, m8, m9 and ml0 are independently either 0
or 1; with the
provision that when m3 is 0, then ml is 0, and when m7 is 0 then either m1-5
are 0 and m8 and m9
are 1 forming a Ma2Ma2 -disaccharide, or both m8 and m9 are 0;
y is anomeric linkage structure a and/or 0 or linkage from derivatized
anomeric carbon, and
R2 is reducing end hydroxyl or chemical reducing end derivative
and x is linkage position 3 or 6 or both 3 and 6 forming branched structure,
{} indicates a branch in the structure.
The invention is further directed to terminal Ma2-containing glycans containg
at least one Ma2-
group and preferably Ma2-group on each branch so that ml and at least one of
m8 or m9 is 1. The
invention is further directed to terminal Ma3 and/or Ma6-epitopes without
terminal Ma2-groups,
when all ml, m8 and m9 are 1.
The invention is further directed in a preferred embodiment to the terminal
epitopes linked to a M(3-
residue and for application directed to larger epitopes. The invention is
especially directed to
M(34GN-comprising reducing end terminal epitopes.
The preferred terminal epitopes comprise typically 2-5 monosaccharide residues
in a linear chain.
According to the invention short epitopes comprising at least 2 monosaccharide
residues can be
recognized under suitable background conditions and the invention is
specifically directed to
epitopes comprising 2 to 4 monosaccharide units and more preferably 2-3
monosaccharide units,
even more preferred epitopes include linear disaccharide units and/or branched
trisaccharide non-
reducing residue with natural anomeric linkage structures at reducing end. The
shorter epitopes may
be preferred for specific applications due to practical reasons including
effective production of
control molecules for potential binding reagents aimed for recognition of the
structures.
The shorter epitopes such as Ma2M is often more abundant on target cell
surface as it is present on
multiple arms of several common structures according to the invention.
Preferred disaccharide epitopes include
Mana2Man, Mana3Man, Mana6Man, and more preferred anomeric forms Mana2Mana,
Mana3Man(3, Mana6Man(3, Mana3Mana and Mana6Mana.
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Preferred branched trisaccharides include Mana3(Mana6)Man, Mana3(Man(x6)Man(3,
and
Mana3(Mana6)Mana.
The invention is specifically directed to the specific recognition of non-
reducing terminal Man(x2-
structures especially in context of high-mannose structures.
The invention is specifically directed to following linear terminal mannose
epitopes:
a) preferred terminal Mana2-epitopes including following oligosaccharide
sequences:
Mana2Man,
Mana2Mana,
Mana2Mana2Man, Mana2Mana3Man, Mana2Mana6Man,
Mana2Mana2Mana, Mana2Mana3Man(3, Mana2Mana6Mana,
Mana2Mana2Mana3Man, Mana2Mana3Mana6Man, Mana2Mana6Mana6Man
Mana2Mana2Mana3Man(3, Mana2Mana3Mana6Man(3, Mana2Mana6Mana6Man(3;
The invention is further directed to recognition of and methods directed to
non-reducing end
terminal Man(x3- and/or Man(x6-comprising target structures, which are
characteristic features of
specifically important low-mannose glycans according to the invention. The
preferred structural
groups include linear epitopes according to b) and branched epitopes according
to the c3) especially
depending on the status of the target material.
b) preferred terminal Mana3- and/or Mana6-epitopes including following
oligosaccharide
sequences:
Mana3Man, Mana6Man, Mana3Man(3, Mana6Man(3, Mana3Mana, Mana6Mana,
Mana3Mana6Man, Mana6Mana6Man, Mana3Mana6Man(3, Mana6Man(x6Man(3
and to following:
c) branched terminal mannose epitopes are preferred as characteristic
structures of especially high-
mannose structures (cl and c2) and low-mannose structures (c3), the preferred
branched epitopes
including:
cl) branched terminal Mana2-epitopes
Mana2Mana3(Mana2Mana6)Man, Mana2Mana3(Mana2Mana6)Mana,
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Mana2Mana3(Mana2Mana6)Mana6Man, Mana2Mana3(Mana2Mana6)Mana6Man(3,
Mana2Mana3 (Mana2Mana6)Mana6(Mana2Man(x3)Man,
Mana2Mana3 (Mana2Mana6)Mana6(Mana2Mana2Mana3)Man,
Mana2Mana3 (Mana2Mana6)Mana6(Mana2Mana3)Man(3
Mana2Mana3 (Mana2Mana6)Mana6(ManaMana2Mana3)Man(3
c2) branched terminal Mana2- and Mana3 or Mana6-epitopes
according to formula when ml and/or m8 and/m9 is 1 and the molecule comprise
at least one
nonreducing end terminal Mana3 or Mana6-epitope
c3) branched terminal Mana3 or Mana6-epitopes
Mana3(Mana6)Man, Mana3(Mana6)Man(3, Mana3(Mana6)Mana,
Mana3(Mana6)Mana6Man, Mana3(Mana6)Mana6Man(3,
Mana3(Mana6)Mana6(Man(x3)Man, Mana3(Mana6)Mana6(Mana3)Man(3
The present invention is further directed to increase the selectivity and
sensitivity in recognition of
target glycans by combining recognition methods for terminal Mana2 and Mana3
and/or Mana6-
comprising structures. Such methods would be especially useful in context of
cell material
according to the invention comprising both high-mannose and low-mannose
glycans.
Complex type N-glycans
According to the present invention, complex-type structures are preferentially
identified by mass
spectrometry, preferentially based on characteristic monosaccharide
compositions, wherein
HexNAc>4 and Hex>3. In a more preferred embodiment of the present invention,
4<HexNAc<20
and 3<Hex<21, and in an even more preferred embodiment of the present
invention, 4<HexNAc<10
and 3<Hex<I 1. The complex-type structures are further preferentially
identified by sensitivity to
endoglycosidase digestion, preferentially N-glycosidase F detachment from
glycoproteins. The
complex-type structures are further preferentially identified in NMR
spectroscopy based on
characteristic resonances of the Mana3(Mana6)Man(34G1cNAc(34G1cNAc N-glycan
core structure
and GIcNAc residues attached to the Mana3 and/or Mana6 residues.
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Beside Mannose-type glycans the preferred N-linked glycomes include G1cNAc(32-
type glycans
including Complex type glycans comprising only G1cNAc(32-branches and Hydrid
type glycan
comprising both Mannose-type branch and G1cNAc(32-branch.
G1cNAc02-type glycans
The invention revealed GlcNAc(32Man structures in the glycomes according to
the invention.
Preferably GlcNAc(32Man-structures comprise one or several of GlcNAc(32Mana -
structures, more
preferably G1cNAc(32Mana3- or GlcNAc(32Mana6-structure.
The Complex type glycans of the invention comprise preferably two
G1cNAc(32Mana structures,
which are preferably GlcNAc02Mana3 and GlcNAc(32Mana6. The Hybrid type glycans
comprise
preferably G1cNAc(32Mana3-structure.
The present invention is directed to at least one of natural oligosaccharide
sequence structures and
structures truncated from the reducing end of the N-glycan according to
the Formul COl (also referred as GN(32):
[RiGN(32]nl [Ma3]n2{[R3]n3 [GN(32]n4Ma6}n5M(34GNXyRz,
with optionally one or two or three additional branches according to formula
[RXGN(3z]nX linked to Ma6-, Ma3-, or M04, and RX may be different in each
branch
wherein nl, n2, n3, n4, n5 and nx, are either 0 or 1, independently,
with the provision that when n2 is 0 then nl is 0 and when n3 is 1 and/or n4
is 1 then n5 is also 1,
and at least nl or n4 is 1, or n3 is 1;
when n4 is 0 and n3 is 1 then R3 is a mannose type substituent or nothing and
wherein X is a glycosidically linked disaccharide epitope (34(Fuca6)nGN,
wherein n is 0 or 1, or X
is nothing and
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
Ri, RX and R3 indicate independently one, two or three natural substituents
linked to the core
structure,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-glycoside
derivative such as asparagine N-glycosides including asparagines N-glycoside
amino acids and/or
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peptides derived from protein; [] indicate groups either present or absent in
a linear sequence, and {
}indicates branching which may be also present or absent.
Elongation of G1cNAc(32-type structures forming complex/hydrid type structures
The substituents Ri, RX and R3 may form elongated structures. In the elongated
structures Ri, and RX
represent substituents of G1cNAc (GN) and R3 is either substituent of G1cNAc
or when n4 is 0 and
n3 is 1 then R3 is a mannose type substituent linked to Mana6-branch forming a
Hybrid type
structure. The substituents of GN are monosaccharide Gal, Ga1NAc, or Fuc
and/or acidic residue
such as sialic acid or sulfate or phosphate ester.
G1cNAc or GN may be elongated to N-acetyllactosaminyl also marked as Gal(3GN
or di-N-
acetyllactosdiaminyl Ga1NAc(3G1cNAc, preferably Ga1NAc(34G1cNAc. LN02M can be
further
elongated and/or branched with one or several other monosaccharide residues
such as galactose,
fucose, SA or LN-unit(s) which may be further substituted by SAa-strutures,
and/or Ma6 residue and/or Ma3 residue can be further substituted by one or two
(36-, and/or (34-
linked additional branches according to the formula;
and/or either of Ma6 residue or Ma3 residue may be absent;
and/or Ma6- residue can be additionally substituted by other Mana units to
form a hybrid type
structures;
and/or Man(34 can be further substituted by GN04,
and/or SA may include natural substituents of sialic acid and/or it may be
substituted by other SA-
residues preferably by a8- or a9-linkages.
The SAa-groups are linked to either 3- or 6- position of neighboring Gal
residue or on 6-position of
G1cNAc, preferably 3- or 6- position of neighboring Gal residue. In separately
preferred
embodiments the invention is directed to structures comprising solely 3-
linked SA or 6- linked SA,
or mixtures thereof.
Preferred Complex type structures
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Incomplete monoantennarv N-glycans
The present invention revealed incomplete Complex monoantennary N-glycans,
which are unusual
and useful for characterization of glycomes according to the invention. The
most of the incomplete
monoantennary structures indicate potential degradation of biantennary N-
glycan structures and are
thus preferred as indicators of cellular status. The incomplete Complex type
monoantennary glycans
comprise only one GN02-structure.
The invention is specifically directed to structures according to the Formula
COl or Formula GNb2
above when only nl is 1 or n4 is 1 and mixtures of such structures.
The preferred mixtures comprise at least one monoantennary complex type
glycans
A) with a single branch likely from a degradative biosynthetic process:
RiGN(32Ma3(34GNXyR2
R3GN(32Ma6M(34GNXyR2 and
B) with two branches comprising mannose branches
Bl) RjGN(32Ma3{Ma6}õ5M(34GNXyR2
B2) Ma3{R3GN(32Ma6}n5M(34GNXyR2
The structure B2 is preferred over A structures as product of degradative
biosynthesis, it is
especially preferred in context of lower degradation of Mana3-structures. The
structure Bl is useful
for indication of either degradative biosynthesis or delay of biosynthetic
process.
Biantennary and multiantennary structures
The inventors revealed a maj or group of biantennary and multiantennary N-
glycans from cells
according to the invention. The preferred biantennary and multiantennary
structures comprise two
GN02 structures. These are preferred as an additional characteristic group of
glycomes according to
the invention and are represented according to the Formula C02:
RiGN(32Ma3 {R3GN(32Ma6}M(34GNXyRz
with optionally one or two or three additional branches according to formula
[RXGN(3z]nX linked to Ma6-, Ma3-, or M04 and RX may be different in each
branch
wherein nx is either 0 or 1,
and other variables are according to the Formula CO1.
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Preferred biantennary structure
A biantennary structure comprising two teiminal GN(3-epitopes is preferred as
a potential indicator
of degradative biosynthesis and/or delay of biosynthetic process. The more
preferred structures are
according to the Formula C02 when R1 and R3 are nothing.
Elongated structures
The invention revealed specific elongated complex type glycans comprising Gal
and/or GaINAc-
structures and elongated variants thereof. Such structures are especially
preferred as informative
structures because the terminal epitopes include multiple informative
modifications of lactosamine
type, which characterize cell types according to the invention.
The present invention is directed to at least one of natural oligosaccharide
sequence structure or
group of structures and corresponding structure(s) truncated from the reducing
end of the N-glycan
according to the Formula C03:
[RiGal[NAc]oz(3z2]oiGN(32Ma3 {[RiGal[NAc]o4(3z2]o3GN(32Ma6}M(34GNXyRz,
with optionally one or two or three additional branches according to formula
[RXGN(3z1]nX linked to Ma6-, Ma3-, or M(34 and RX may be different in each
branch
wherein nx, ol, o2, o3, and o4 are either 0 or 1, independently,
with the provision that at least ol or o3 is 1, in a preferred embodiment both
are 1;
z2 is linkage position to GN being 3 or 4, in a preferred embodiment 4;
zl is linkage position of the additional branches;
Rl, Rx and R3 indicate one or two a N-acetyllactosamine type elongation groups
or nothing,
{} and O indicates branching which may be also present or absent,
other variables are as described in Formula GNb2..
Galactosylated structures
The inventors characterized useful structures especially directed to
digalactosylated structure
Gal(3zGN(32Ma3 {Gal(3zGN(32Ma6}M(34GNXyRz,
and monogalactosylated structures:
Gal(3zGN(32Ma3 {GN(32Ma6}M(34GNXyRz,
GN(32Ma3{Gal(3zGN(32Ma6}M(34GNXyRz,
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and/or elongated variants thereof preferred for carrying additional
characteristic terminal structures
useful for characterization of glycan materials
RiGal(3zGN(32Ma3 {R3Ga1(3zGN(32Ma6}M(34GNXyRz
R1Ga1(3zGN(32Ma3{GN(32Ma6}M(34GNXyRz, and
GN(32Ma3 {R3Gal(3zGN(32Ma6}M(34GNXyR2.
Preferred elongated materials include structures wherein Ri is a sialic acid,
more preferably
NeuNAc or NeuGc.
LacdiNAc-structure comprising N-glycans
The present invention revealed for the first time LacdiNAc, GaLNAc(3G1cNAc
structures from the
cell according to the invention. Preferred N-glycan lacdiNAc structures are
included in structures
according to the Formula CO 1, when at least one the variable o2 and o4 is 1.
The major acidic glycan types
The acidic glycomes mean glycomes comprising at least one acidic
monosaccharide residue such as
sialic acids (especially NeuNAc and NeuGc) forming sialylated glycome, HexA
(especially G1cA,
glucuronic acid) and/or acid modification groups such as phosphate and/or
sulphate esters.
According to the present invention, presence of sulphate and/or phosphate
ester (SP) groups in
acidic glycan structures is preferentially indicated by characteristic
monosaccharide compositions
containing one or more SP groups. The preferred compositions containing SP
groups include those
formed by adding one or more SP groups into non-SP group containing glycan
compositions, while
the most preferential compositions containing SP groups according to the
present invention are
selected from the compositions described in the acidic N-glycan fraction
glycan group Tables of the
present invention. The presence of phosphate and/or sulphate ester groups in
acidic glycan
structures is preferentially further indicated by the characteristic fragments
observed in
fragmentation mass spectrometry corresponding to loss of one or more SP
groups, the insensitivity
of the glycans carrying SP groups to sialidase digestion. The presence of
phosphate and/or sulphate
ester groups in acidic glycan structures is preferentially also indicated in
positive ion mode mass
spectrometry by the tendency of such glycans to form salts such as sodium
salts as described in the
Examples of the present invention. Sulphate and phosphate ester groups are
further preferentially
identified based on their sensitivity to specific sulphatase and phosphatase
enzyme treatments,
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respectively, and/or specific complexes they form with cationic probes in
analytical techniques such
as mass spectrometry.
Sialylated Complex N-glycan glycomes
The present invention is directed to at least one of natural oligosaccharide
sequence structures and
structures truncated from the reducing end of the N-glycan according to
the Formula
[{SAa3/6}SiLN(32]r1Ma3{({SA(x3/6}s2LN(32)r2Ma6}r8
{M[(34GN[(34{Fuca6}r3GNr4]r5}r6
(I)
with optionally one or two or three additional branches according to formula
{SAa3/6}s3LN(3, (IIb)
wherein rl, r2, r3, r4, r5, r6, r7 and r8 are either 0 or 1, independently,
wherein s1, s2 and s3 are either 0 or 1, independently,
with the provision that at least rl is 1 or r2 is 1, and at least one of sl,
s2 or s3 is 1.
LN is N-acetyllactosaminyl also marked as Gal(3GN or di-N-acetyllactosdiaminyl
GaLNAc(3G1cNAc preferably Ga1NAc(34G1cNAc, GN is G1cNAc, M is mannosyl-,
with the provision that LN(32M or GN(32M can be further elongated and/or
branched with one or
several other monosaccharide residues such as galactose, fucose, SA or LN-
unit(s) which may be
further substituted by SAa-strutures,
and/or one LN(3 can be truncated to GN(3
and/or Ma6 residue and/or Ma3 residue can be further substituted by one or two
(36-, and/or (34-
linked additional branches according to the formula,
and/or either of Ma6 residue or Ma3 residue may be absent;
and/or Ma6- residue can be additionally substituted by other Mana units to
form a hybrid type
structures
and/or Man(34 can be further substituted by GN04,
and/or SA may include natural substituents of sialic acid and/or it may be
substituted by other SA-
residues preferably by a8- or 0-linkages.
O, {}, [] and [] indicate groups either present or absent in a linear
sequence. {}indicates
branching which may be also present or absent.
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The SAa-groups are linked to either 3- or 6- position of neighboring Gal
residue or on 6-position of
G1cNAc, preferably 3- or 6- position of neighboring Gal residue. In separately
preferred
embodiments the invention is directed structures comprising solely 3- linked
SA or 6- linked SA, or
mixtures thereof. In a preferred embodiment the invention is directed to
glycans wherein r6 is 1 and
r5 is 0, corresponding to N-glycans lacking the reducing end GIcNAc structure.
The LN unit with its various substituents can be represented in a preferred
general embodiment by
the formula:
[Gal(NAc)nia3]n2{Fuca2}n3Gal(NAc)n4(33/4{Fuca4/3}nsGlcNAc(3
wherein nl, n2, n3, n4, and n5 are independently either 1 or 0,
with the provision that the substituents defined by n2 and n3 are alternative
to the presence of SA at
the non-reducing end terminal structure;
the reducing end G1cNAc -unit can be further 03- and/or 06-linked to another
similar LN-structure
forming a poly-N-acetyllactosamine structure with the provision that for this
LN-unit n2, n3 and n4
are 0,
the Gal(NAc)(3 and G1cNAc(3 units can be ester linked a sulphate ester group;
O and [] indicate groups either present or absent in a linear sequence;
{}indicates branching which
may be also present or absent.
LN unit is preferably Ga1(34GN and/or Gal(33GN. The inventors revealed that
hESCs can express
both types of N-acetyllactosamine, and therefore the invention is especially
directed to mixtures of
both structures. Furthermore, the invention is directed to special relatively
rare type 1 N-
acetyllactosamines, Gal(33GN, without any non-reducing end/site modification,
also called lewis c-
structures, and substituted derivatives thereof, as novel markers of hESCs.
Hybrid type structures
According to the present invention, hybrid-type or monoantennary structures
are preferentially
identified by mass spectrometry, preferentially based on characteristic
monosaccharide
compositions, wherein HexNAc=3 and Hex>2. In a more preferred embodiment of
the present
invention 2<Hex<11, and in an even more preferred embodiment of the present
invention 2<Hex<9.
The hybrid-type structures are further preferentially identified by
sensitivity to exoglycosidase
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digestion, preferentially a-mannosidase digestion when the structures contain
non-reducing terminal
a-mannose residues and Hex>3, or even more preferably when Hex>4, and to
endoglycosidase
digestion, preferentially N-glycosidase F detachment from glycoproteins. The
hybrid-type
structures are further preferentially identified in NMR spectroscopy based on
characteristic
resonances of the Mana3(Mana6)Man(34G1cNAc(34G1cNAc N-glycan core structure, a
G1cNAc(3
residue attached to a Mana residue in the N-glycan core, and the presence of
characteristic
resonances of non-reducing terminal a-mannose residue or residues.
The monoantennary structures are further preferentially identified by
insensitivity to a-mannosidase
digestion and by sensitivity to endoglycosidase digestion, preferentially N-
glycosidase F
detachment from glycoproteins. The monoantennary structures are further
preferentially identified
in NMR spectroscopy based on characteristic resonances of the
Mana3Man(34G1cNAc(34GlcNAc
N-glycan core structure, a G1cNAc(3 residue attached to a Mana residue in the
N-glycan core, and
the absence of characteristic resonances of further non-reducing terminal a-
mannose residues apart
from those arising from a terminal a-mannose residue present in a ManaMan(3
sequence of the N-
glycan core.
The invention is further directed to the N-glycans when these comprise hybrid
type structures
according to the Formula HY1:
RIGN(32Ma3 {[R3]n3Ma6}M(34GNXyRz,
wherein n3, is either 0 or 1, independently,
and wherein X is glycosidically linked disaccharide epitope 04(Fuca6)nGN,
wherein n is 0 or 1, or
X is nothing and
y is anomeric linkage structure a and/or 0 or linkage from derivatized
anomeric carbon, and
Ri indicate nothing or substituent or substituents linked to G1cNAc,
R3 indicates nothing or Mannose-substituent(s) linked to mannose residue, so
that each of Ri, and
R3 may correspond to one, two or three, more preferably one or two, and most
preferably at least
one natural substituents linked to the core structure,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-glycoside
derivative such as asparagine N-glycosides including asparagines N-glycoside
amino acids and/or
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peptides derived from protein; [] indicate groups either present or absent in
a linear sequence, and {
}indicates branching which may be also present or absent.
Preferred hybrid type structures
The preferred hydrid type structures include one or two additional mannose
residues on the
preferred core stucture.
Formula HY2
R1GN(32Ma3 {[Ma3]ml([M(x6])mzMa6}M(34GNXyRz,
wherein and ml and m2 are either 0 or 1, independently,
{} and O indicates branching which may be also present or absent,
other variables are as described in Formula HY1.
Furthermore the invention is directed to structures comprising additional
lactosamine type
structures on GN(32-branch. The preferred lactosamine type elongation
structures includes N-
acetyllactosamines and derivatives, galactose, Ga1NAc, GIcNAc, sialic acid and
fucose.
Preferred structures according to the formula HY2 include:
Structures containing non-reducing end terminal GIcNAc as a specific preferred
group of glycans
GN02Ma3 {Ma3Ma6}M(34GNXyR2,
GN(32Ma3 {Ma6Ma6}M(34GNXyR2,
GN(32Ma3 {Ma3 (Ma6)Ma6}M(34GNXyRz,
and/or elongated variants thereof
R1GN(32Ma3 {Ma3Ma6}M(34GNXyRz,
RiGN(32Ma3 {Ma6Ma6}M(34GNXyR2,
RiGN(32Ma3 {Ma3(Ma6)Ma6}M(34GNXyR2,
Formula HY3
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[RiGal[NAc]oz(3z]oiGN(32Ma3{[Ma3],,i[(Ma6)]mzMa6}n5M(34GNXyRz,
wherein n5, ml, m2, ol and o2 are either 0 or 1, independently,
z is linkage position to GN being 3 or 4, in a preferred embodiment 4,
Ri indicates one or two a N-acetyllactosamine type elongation groups or
nothing,
{} and O indicates branching which may be also present or absent,
other variables are as described in Formula HY1.
Preferred structures according to the formula HY3 include especially
structures containing non-reducing end terminal Gal(3, preferably Gal(33/4
forming a terminal N-
acetyllactosamine structure. These are preferred as a special group of Hybrid
type structures,
preferred as a group of specific value in characterization of balance of
Complex N-glycan glycome
and High mannose glycome:
Gal(3zGN(32Ma3{Ma3M(x6}M(34GNXyRz, Gal(3zGN(32Ma3{M(x6Ma6}M(34GNXyRz,
Gal(3zGN(32Ma3 {Ma3(Ma6)Ma6}M(34GNXyRz,
and/or elongated variants thereof preferred for carrying additional
characteristic terminal structures
useful for characterization of glycan materials
RiGal(3zGN(32Ma3{Ma3Ma6{M(34GNXyRz, RiGal(3zGN(32Ma3{Ma6Ma6}M(34GNXyRz,
RiGal(3zGN(32Ma3{Ma3(Ma6)Ma6}M(34GNXyR2. Preferred elongated materials include
structures wherein Ri is a sialic acid, more preferably NeuNAc or NeuGc.
Structures associated with nondifferentiated hESC
The Tables 1 and 2 show specific structure groups with specific monosaccharide
compositions
associated with the differentiation status of human embryonic stem cells.
The structures present in higher amount in hESCs than in corresponding
differentiated cells
The invention revealed novel structures present in higher amounts in hESCs
than in corresponding
differentiated cells.
The preferred hESC enriched glycan groups are represented by groups hESC-i to
hESC-ix,
corresponding to several types of N-glycans. The glycans are preferred in the
order from hESC-i to
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hESC-ix, based on the relative specificity for the non-differentiated hESCs,
the differences in
expression are shown in Tables 1 and 2. The glycans are grouped based on
similar composition and
similar structures present to group comprising Complex type N-glycans other
preferred glycan
groups,
Complex type 21vcans
hESC-i, Biantennary-size complex-type N-glycans
The highest specific expression in hESCs was revealed for a specific group of
biantennary complex
type N-glycan structures. This group includes neutral glycans including H5N4F
1, H5N4F2,
H5N4F3; and sialylated glycans G2H5N4, G1H5N4, S1H5N4F2, G1H5N4F1, S1G1H5N4,
S1H5N4F3, S2H5N4F1, S1H5N4, and S1H5N4F1.
Preferred structural sub,groups of the biantennar,y complex type glycans
include Neutral fucosylate
glycans and NeuAc comprising fucosylated glycans and glycans comprising NeuGc.
Neutral fucosylated glycans
The group of neutral glycans forms a homogenous group with typical composition
of biantennary
N-glycans and one, two or three fucose residues. This group shares a common
composition:
H5N4Fq
Wherein
q is an integer being 1, 2 or 3.
The preferred structures in this group include
[Fuca]mGalPGN02Mana3([Fuca]nGa1PGN02Mana6)Manp4GNP4(Fuca6)GN,
wherein m and n are 0 or 1, GN is G1cNAc. The structures are preferably core
fucosylated, when
there is only one fucose. (The core fucosylation was revealed by NMR-analysis
of the hESC
glycans.) The fucose residues at the antennae (branches) are preferably either
Fuca2-structures
linked to Gal or Fuca3/4-structures, preferably Fuca3, linked to G1cNAc of the
terminal N-
acetyllactosamines.
Preferred fucosylated terminal epitopes [Fuca]Gal(3G1cNAcP2Mana
Prefered Lewis x epitopes
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The preferred terminal epitopes, which can be recognized from hESCs by
specific binder
molecules, include Lewis x, Gal(34(Fuc(x3)GIcNAc(3, more preferably
Gal(34(Fuca3)G1cNAc(32Mana, based on binding of specific Lewis x recognizing
monoclonal
antibody.
The invention is further directed to the recognition of the Lewis x structure
as a specific preferred
arm of N-glycan selected from the group Gal(34(Fuc(x3)G1cNAc(32Mana3Man(3
(Lex(32Mana3-
arm) and/or Gal(34(Fuca3)G1cNAc(32Mana6Man(3 (Lex(32Mana6-arm). The invention
is directed
to selection and development of reagents for the specific fucosylated N-glycan
arms for recognition
of N-glycans on the human embryonic stem cells and derivatives.
The H-antigens on N-glycans includes preferably the epitope
Fuca2Ga1(3G1cNAc(3, preferably H
type I Fuca2Gal(33GlcNAc(3 or H type II structure Fuca2Gal(34GlcNAc(3, more
preferably
Fuca2Ga1(34GIcNAc(3, and most preferably Fuca2Gal(34GlcNAc(32Mana.
The invention is further directed to the recognition of the H type II
structure as a specific preferred
arm of N-glycan selected from the group
Fuca2Ga1(34G1cNAc(32Mana3Man(3 (HLacNAc(32Mana3-arm) and/or
Fuca2Ga1(34GIcNAc(32Mana6Man(3 (HLacNAc(32Mana6-arm). The invention is
directed to
selection and development of reagents for the specific fucosylated N-glycan
arms for recognition of
N-glycans on the human embryonic stem cells and derivatives.
Preferred neutral difucosylated structures include glycans comprising core
fucose and the terminal
Lewis x or H-antigen on either arm of the biantennary N-glycan according to
the formulae:
Gal(34(Fuca3)GN(32Mana3/6(Gal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN, and/or
Fuca2Ga1(3GN(32Mana3/6(Gal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN.
Preferred neutral trifucosylated structures includes glycans comprising core
fucose and the terminal
Lewis x or H-antigen on either arm of the biantennary N-glycan according to
the formulae:
Gal(34(Fuca3)GN(32Mana3/6([Fuca]Gal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN, and/or
Fuca2Ga1(3GN(32Mana3/6([Fuca]Gal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN,
Wherein the molecules comprise two H-structures, Lewis x in one arm and H-
structure in the the
other arm or two Lewis x structures:
Fuca2Ga1(3GN(32Mana3 (Fuca2Ga1(3GN(32Mana6)Man(34GN(34(Fuca6)GN,
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Gal(34(Fuca3)GN(32Mana3/6(Fuca2Ga1(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN
Gal(34(Fuca3)GN(32Mana3(Gal(34(Fuca3)GN(32Mana6)Man(34GN(34(Fuca6)GN,
Or molecules comprising Lewis y on one arm:
Fuca2Ga1(34(Fuca3)GN(32Mana3/6(Gal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN
NeuAc comprising fucosylated glycans
The sialylated glycans include NeuAc comprising fucosylated glycans with
formulae: S1H5N4F2,
S1H5N4F3, S2H5N4F1, SIH5N4, and S1H5N4F1. This group shares composition:
SkH5N4Fq
Wherein
k is an integer being 1 or 2
q is an integer from 0 to 3.
The group comprises monosialylated glycans with all levels of fucosylation and
disialylated glycan
with single fucose. The preferred subgroups of this category include low
fucosylation level glycans
comprising no or one fucose residue (low fucosylation) and glycans with two or
three fucose
residues.
Preferred biantennaNy structures with low fucosylation
The preferred biantennary structures according to the invention include
structures according to the
Formula:
[NeuAca]o_iGa1PGNp2Mana3([NeuAca]o_iGa1PGN02Mana6)Manp4GN04(Fuca6)o_iGN,
The Gal(3G1cNAc structures are preferably Gal(34G1cNAc-structures (type II N-
acetyllactosamine
antennae). The presence of type 2 structures was revealed by specific (34-
linkage cleaving
galactosidase (D. pneumoniae).
In a preferred embodiment the sialic acid is NeuAca6- and the glycan comprises
the NeuAc linked
to Mana3-arm of the molecule. The assignment is based on the presence of a6-
linked sialic acid
revealed by specific sialidase digestion and the known branch specificity of
the a6-sialyltransferase
(ST6GaI1).
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NeuAca6Ga1(3GN(32Mana3([NeuAca]o_iGal(3GN(32Mana6)Man(34GN(34(Fuca6)o_iGN,
more
preferably type II structures:
NeuAca6Ga1(34GN(32Mana3([NeuAca]o_iGal(34GN(32Man(Y,6)Man(34GN(34(Fuca6)o_iGN.
The invention thus revealed preferred terminal epitopes, NeuAca6Gal(3GN,
NeuAca6Ga1(3GN(32Man, NeuAca6Gal(3GN(32Mana3, to be recognized by specific
binder
molecules. It is realized that higher specificity preferred for application in
context of similar
structures can be obtained by using binder recognizing longer epitopes and
thus differentiating e.g.
between N-glycans and other glycan types in context of the terminal epitopes.
Preferred di ucosylated and sialylated structures
Preferred difucosylated sialylated structures include structures, wherein the
one fucose is in the core
of the N-glycan and
a) one fucose on one arm of the molecule, and sialic acid is on the other arm
(antenna of the
molecule and the fucose is in Lewis x or H-structure:
Gal(34(Fuca3)GN(32Mana3/6(NeuNAcaGal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN,
and/or
Fuca2Ga1(3GN(32Mana3/6(NeuNAcaGal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN, and when
the
sialic acid is a6-linked preferred antennary structures contain preferably the
sialyl-lactosamine on
0-linked arm of the molecule according to formula:
Ga1P4(Fuca3)GN(32Mana6(NeuNAca6Galp4GN(32Mana3)Manp4GNP4(Fuca6)GN,
and/or
Fuca2GalPGNp2Mana6(NeuNAc(x6Gal(34GNp2Mana3)Manp4GN04(Fuca6)GN.
It is realized that the structures, wherein the sialic acid and fucose are on
different arms of the
molecules can be recognized as characteristic specific epitopes.
b) Fucose and NeuAc are on the same arm in a structure:
NeuNAca3Ga1(33/4(Fuca4/3)GN(32Mana3/6(Gal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN,
and more preferably sialylated and fucosylated sialyl-Lewis x structures are
preferred as a
characteristic and bioactive structures:
NeuNAca3Ga1(34(Fuca3)GN(32Mana3/6(Gal(34GN(32Mana6/3)Man(34GN(34(Fuca6)GN.
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Preferred sialylated trifucosylated structures
Preferred sialylated trifucosylated structures include glycans comprising core
fucose and the
terminal sialyl-Lewis x or sialyl-Lewis a, preferably sialyl-Lewis x due to
relatively large presence
of type 2 lactosamines, or Lewis y on either arm of the biantennary N-glycan
according to the
formulae:
NeuNAca3Ga1(34(Fuca3)GN(32Mana3/6([Fuca]Gal(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN,
and/or
Fuca2Ga1(34(Fuca3)GN(32Mana3/6(NeuNAca3/6Ga1(3GN(32Mana6/3)Man(34GN(34(Fuca6)GN
.
NeuNAc is preferably a-linked on the same arm as fucose due to known
biosynthetic preferance.
When the structure comprises NeuNAca6, this is preferably linked to form
NeuNAca6Ga1(34G1cNAc(32Mana3-arm of the molecule.
Glvcans compNising N-glycolylneuraminic acid
The invention is directed to glycans comprising N-glycolylneuraminic acid with
following
compositions G2H5N4, G1H5N4, G1H5N4F1, and S1G1H5N4. The compositions form a
group of
compositions with composition:
GmSkH5N4Fq
wherein
m is an integer being 1 or 2,
k is an integer being 0 or 1, and
q is an integer being 0 or 1.
The invention is further directed to the structures according to the formula:
[NeuXa]o_iGal(3GN(32Mana3/6([NeuXa]o_iGal(3GN(32Mana6/3)Man(34GN(34(Fuca6)o_iGN
,
wherein X is Gc or Ac, and the sialic acids are linked by a3- and/or a6-
linkages.
It is further realized that it is useful to analyze the NeuGc comprising
structures in context of
contamination by animal protein and or animal derived NeuGc-monosaccharide or
glycoconjugate
comprising material.
hESC-ii, Complex-fucosylated N-glycans
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The invention is further directed to following neutral glycans including
H5N4F2, H5N4F3,
H4N5F3; and sialylated glycans including S 1 H7N6F2, S 1 H7N6F3, S 1 H5N4F2, S
1 H6N5F2,
SIH6N4F2, S1H5N4F3, SIH4N5F2, S2H6N5F2, S1H6N5F3;
preferentially with al,2-, al,3-, and/or al,4-linked fucose residues within
the N-acetyllactosamine
antenna sequence Gal(33/4G1cNAc forming H and/or Lewis antigens, more
preferentially type II N-
acetyllactosamine (Ga1P4G1cNAc) forming H type 2, Lewis x, sialyl Lewis x,
and/or Lewis y
antigens.
LacdiNAc comprisinz S1/0H4N5F2/3-structures
In a preferred embodiment, the invention is directed to analysis of structure
of preferred N-glycans
with S1/0H4N5F2/3 structures, when the composition comprises biantennary N-
glycan type
structures with terminal LacdiNAc structure. The LacdiNAc epitope has
structure
GaLNAc(3G1cNAc, preferably GaLNAc(34G1cNAc and preferred sialylated LacdiNAc
epitope has the
structure NeuAca6GalNAc(34G1cNAc, based on the known mammalian glycan
structure
information. Based on biosynthetic knowledge the a6-sialylated structure
likely not comprises
fucose. The preferred sialyl-lactosamine structures includes
NeuAca3/6Ga1(34G1cNAc. The
presence of lacdinac structures was revealed by N-acetylhexosaminidase and N-
acetylglucosaminidase digestions.
The invention is especially directed to the composition with terminal Lewis x
epitope and a
sialylated LacdiNAc epitope according to the Formula:
Gal(34(Fuca3)GNp2Mana3/6(NeuAc(x6GalNAc(34GN(32Mana6/3)Man(34G1cNAc(34(Fuca6)GN
.
The invention is especially directed to the composition with terminal Lewis x
epitope and a
fucosylated LacdiNAc epitope according to the Formula:
Gal(34(Fuca3)GNp2Mana3/6(Ga1NAc(34(Fuca3)GN(32Mana6/3)Man(34G1cNAc(34(Fuca6)GN,
and/or structure with Lewis y and LacdiNAc:
Fuca2Gal(34(Fuca3)GN(32Mana3/6(Ga1NAc(34GN(32Mana6/3)Man(34GIcNAc(34(Fuca6)GN.
Multiple N-acetyllactosamine comprisin,- structures
The invention is further directed to multiple (more than 2) N-
acetyllactosamine comprising N-
glycan structures according to the formulae: S1H7N6F2, SIH7N6F3, S1H6N5F2,
S2H6N5F2, and
SIH6N5F3.
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Preferred triantennary glycans
The invention is especially directed to triantennary N-glycans having
compositions SIH6N5F2,
S2H6N5F2, and S1H6N5F3. Presence of triantennary structures was revealed by
specific
galactosidase digestions. A preferred type of triantennary N-glycans includes
one synthesized by
Mgat3. The triantennary N-glycan comprises in a preferred embodiment a core
fucose residue. The
preferred terminal epitopes include Lewis x, sialyl-Lewis x, H- and Lewis y
antigens as described
above for biantennary N-glycans.
Preferred tetraantennary and/oN polylactosamine structures
The invention is further directed to monosaccharide compositions and glycan
corresponding to
monosaccharide compositions S1H7N6F2, and SIH7N6F3, which were assigned to
correspond to
tetra-antennary and/or poly-N-acetyllactosamine epitope comprising N-glycans
such as ones with
terminal Gal(3GlcNAc(33Gal(3GlcNAc(3-, more preferably type 2 structures
Gal(34GlcNAc(33Ga1(34G1cNAc(3-.
hESC-vi, Large complex-type N-glycans
The preferred group includes neutral glycans with compositions H6N5, and H6N5F
1.
The preferred structures in this group include:
triantennary N-glycans, in a preferred embodiment the triantennary N-glycan
comprises (31,4-linked
N-acetyllactosamine, preferably linked to Mana6-arm of the N-glycan (mgat4
product N-glycan)
and poly-N-acetyllactosamine elongated biantennary complex-type N-glycans.
hESC-vii, Monoantennary type N-2lycans
The preferred group includes neutral glycans with compositions including H4N3,
and H4N3F1;
And preferentially corresponding to structures:
Gal(3G1cNAc(32Mana3(Mana6)Man(34GIcNAc(34(Fuca6)o_iG1cNAc, more preferentially
with type
II N-acetyllactosamine antennae, wherein galactose residues are (31,4-linked
Gal(34GlcNAc(32Mana3(Mana6)Man(34G1cNAc(34(Fuca6)o_iGlcNAc.
hESC-viii, Terminal HexNAc comulex-type N-glycans
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The preferred group includes neutral glycans having composition H4N5F3; and
sialylated glycans
including S2H4N5F1, and S1H4N5F2.
hESC-ix, Elongated lar2e complex-type N-glycans
The preferred group includes glycans having composition S1H8N7F1, S1H7N6F2,
S1H7N6F3, and
S1H7N6F1;
preferentially including poly-N-acetyllactosamine sequences.
Terminal Mannose N-glycans
High mannose type glycans
hESC-iii, High-mannose type N-glycans, including H6N2, H7N2, H8N2, and
H9N2.The preferred
high Mannose type glycans are according to the formula:
[Ma2]õlMa3 {[Ma2]n3Ma6}Ma6{[Ma2],6[Ma2]n-,Ma.3}M(34GN(34GNyRz
wherein nl, n3, n6, and n7are either independently 0 or 1;
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-glycoside
derivative such as asparagine N-glycosides including aminoacid and/or peptides
derived from
protein;
[] indicates determinant either being present or absent depending on the value
of nl, n3, n6, n7; and
{} indicates a branch in the structure;
M is D-Man, GN is N-acetyl-D-glucosamine., y is anomeric structure or linkage
type, preferably beta to
Asn.
The preferred structures in this group include:
Mana2Mana6(Mana2Mana3)Mana6(Mana2Mana2Mana3)Man(34G1cNAc(34G1eNAc
Mana2Mana6([Mana2]o_iMana3)Mana6([Mana2]o_iMana2Mana3)Man(34G1eNAc(34G1cNAc
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hESC-v, Glucosylated high-mannose type N-glycans, including H10N2, H11N2;
preferentially including:
Mana2Mana6(Mana2Mana3)Mana6([Glca]o_
iGlcaMana2Mana2Mana3)Man(34G1cNAc(34G1cNAc
Specific Low mannose type lzlvcan
hESC-iv, Monomannose N-glycan H1N2;
preferentially including the structure Man(34G1cNAc(34GlcNAc.
Structures and compositions associated with differentiated cell types (EB and
St.3)
The invention revealed novel structures present in higher amount in
differentiated embryonic stem
cells than in corresponding non-differentiated hESCs.
The preferred glycan groups are represented in groups Diff-i to Diff-ix,
corresponding to several
types of N-glycans. The glycans are preferred in the order from Diff-i to Diff-
ix, based on the
relative specificity for the non-differentiated hESCs, the differences in the
expression are shown in
Tables 1 and 2
Terminal Mannnose N-glycans
Preferred terminal Low Mannose N-glycans
Diff-i, Low-mannose type N-glycans,
The preferred low mannose glycans have compositions H2N2, H3N2, and H4N2; and
fucosylated
low-mannose type N-glycans, including H2N2F1, H3N2F1, and H4N2F1.
Several preferred low Man glycans described above can be presented in a
Formula:
[Ma3]n2{[M(x6)]n4}[M(x6]n5{[Ma3]n8}M(34GN(34[{Fuca6}]mGNyRz
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wherein n2, n4, n5, n8, and m are either independently 0 or 1; [] indicates
determinant being either
present or absent depending on the value of n2, n4, n5, n8 and m, {} indicates
a branch in the
structure;
y and R2 are as indicated for Formula M2.
Preferred non-fucosylated Low mannose N-glycans are according to the Formula:
Ma6M(34GN(34GNyR2
Ma3M(34GN(34GNyR2 and
Ma6{Ma3}M(34GN(34GNyR2.
Ma6Ma6{Ma3}M(34GN(34GNyR2
Ma3Ma6{Ma3}M(34GN(34GNyR2
Preferred individual structures of fucosvlated low-mannose kl cy ans
Small fucosylated low-mannose structures are especially unusual among known N-
linked glycans
and form a characteristic glycan group useful for the methods according to the
invention, especially
analysis and/or separation of cells according to the present invention. These
include:
M(34GN(34(Fuc(x6)GNyR2
Ma6M(34GN(34(Fuca6)GNyR2
Ma3M(34GN(34(Fuca6)GNyR2 and
Ma6Ma6{Ma3}M(34GN(34(Fuca6)GNyR2 and
Ma3Ma6{Ma3}M(34GN(34(Fuca6)GNyR2 and
In a specific embodiment the low mannose glycans includes rare structures
based on unusual
mannosidase degradation
Mana2Mana2Mana3Man(34GN(34(Fuca6)o_iGN, Mana2Mana3Man(34GN(34(Fuca6) o_iGN.
High mannose type 2lycans
Diff-ii, Fucosylated high-mannose type N-glycans, including H5N2F1, H6N2F1;
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preferentially including:
Mana6(Mana3)Mana6(Mana3)Man(34G1cNAc(34(Fuca6)G1cNAc; and
[Mana2]o_iMana6([Mana2]o_iMana3)Mana6(Mana3)Man(34G1cNAc(34(Fuca6)G1cNAc
Diff-iii, Small high-mannose type N-glycans, including H5N2, preferably
corresponding to the
structure
Mana6(Mana3)Mana6(Mana3)Man(34G1cNAc(34G1cNAc
Complex type glycans
Diff-iv, Terminal HexNAc N-glycans, including H5N6F2, H3N4, H3N5, H4N4F2,
H4N5F2,
H4N4, H4N5F1, H2N4F1, H3N5F1, and H3N4F1.
The preferred H4H5 structures, H4N5F2 and H4N5F1, include following preferred
structures
comprising LacdiNAc:
[Fuca]n3 {Gal[NAc]ni(3GN(32Mana3(Gal[NAc] 20GN(32Mana6)Man(34GN04(Fuc(x6)n2GN,
wherein nl and n2 are either 0 or 1, so that either nl or n2 is 0 and the
other is 1 and n3 is either 0
or 1. The fucose residue forms preferably Lewis x or fucosylated LacdiNAc
structure
GaLNAc(34(Fuc(x3)GlcNAc.
Diff-v, Hybrid-type N-glycans, including H5N3F1, H5N3, H6N3F1, and H6N3.
The preferred structures in this group are according to the Formula:
[Gal(3]niGlcNAc(32Mana3(Mana3 [Mana6]Mana6)Man(34G1cNAc(34(Fuca6)nzGlcNAc
Wherein nl and n2 are either 0 or 1.
The preferred H5N3 structures are according to the Formula
G1cNAc(32Mana3 (Man(x3 [Mana6]Mana6)Man(34G1cNAc(34(Fuca6)nzGlcNAc
Wherein n2 is either 0 or 1.
The preferred H6N3 structures are according to the Formula
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Gal(3G1cNAc(32Mana3(Mana3[Mana6]Mana6)Man(34G1cNAc(34(Fuca6)nzGleNAc
wherein n2 is either 1 or 0.
Diff-vi, Terminal HexNAc monoantennary N-glycans, including H3N3, H3N3F 1, and
H2N3F 1;
preferentially including:
G1cNAc(32Mana3([Mana6]o_i)Man(34GlcNAc(34(Fuca6)o_iGlcNAc, more preferentially
with type II
N-acetyllactosamine antennae, wherein galactose residues are (31,4-linked.
Diff-vii, H=N type terminal HexNAc N-glycans, including H5N5F1, H5N5, H5N5F3
Terminal HexNAc, especially terminal G1cNAc glycans of this type are described
below in more
detail.
Diff-viii, Elongated hybrid-type N-glycans, including H6N4, H7N4
Gal(3GN(3[ (]niGal(3GN[ )]nz(32Mana3([Mana3]n3[Mana6]n4Man(x6)Man(34GN(34GN
nl, and n2 are both either 0 indicating linear structure or 1 indicating a
branched structure and n3
and n4 is either 0 or 1, so that at least one is 1. More preferably the
structure comprises linear
polylactosamine (both nl and n2 are 0):
Gal(3G1cNAc(3Ga1(3G1cNAc(32Mana3([Mana3]n3[Mana6]n4Mana6)Man(34GlcNAc(34G1cNAc,
preferably comprising a(33-linkage between the lactosamines
Gal(3G1cNAc(33Ga1(3GlcNAc, and
even more preferably type 2 N-acetyllactosamines Gal(34GlcNAc(33Gal(34GlcNAc.
Diff-ix, Complex-fucosylated monoantennary type N-glycans, including H4N3F2;
preferably including:
FucaGal(3G1cNAc(32Mana3([Mana6]o-i)Man(34GlcNAc(34(Fuca6)G1cNAc, preferably
the fucose is
Fuca2linked to Gal, or Fuca3/4 linked to G1cNAc;
more preferentially with type II N-acetyllactosamine antennae:
FucaGal(34GlcNAc(32Mana3([Mana6]o-,)Man(34G1cNAc(34(Fuca6)G1cNAc, even more
preferably
Fuca2Gal(34GlcNAc(32Mana3([Mana6]o_i)Man(34GlcNAc(34(Fuca6)GIcNAc and/or
Gal[34(Fuca3)G1eNAc(32Mana3([Mana6]o-i)Man(34G1cNAc[34(Fuca6)G1cNAc.
Novel Terminal HexNAc N-glycan compositions from stem cells
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The inventors studied human stem cells as shown in EXAMPLE 1. The data
revealed a specific
group of altering glycan structures referred as terminal HexNAc structures as
shown in Table 5. The
figure 1 reveals changes of preferred signals in context of differentiation.
The terminal HexNAc
structures were assigned to include terminal N-acetylglucosamine structures by
cleavage with N-
acetylglucosamidase enzymes. The Example 2 reveals the analysis of changes of
the structures in
multiple types of stem cells, the corresponding expression data is summarized
in Tables 2 and 3,
especially under terminal HexNAc structures.
Preferred N-izlycans according to structural sub,groups with terminal HexNAc
The inventors found that there are differentiation stage specific differences
with regard to terminal
HexNAc containing N-glycans characterized by the formulae: nxeXNac = nxeX >_ 5
and ndHeX > 1
(group I), or: nxeXNA, = nxeX > 5 and naxeX = 0 (group II). The present data
demonstrated that these
glycans were 1) detected in various N-glycan samples isolated from both stem
cells, including
hESC, and cells directly or indirectly differentiated from these cell types;
and 2) overexpressed in
the analyzed differentiated cells when compared to the corresponding stem
cells. There was
independent expression between groups I and group II and therefore, the N-
glycan structure group
determined by the formula nHeXNA, = nxeX > 5 is divided into two independently
expressed
subgroups I and II as described above.
Based on the known specificities of the biosynthetic enzymes synthesizing N-
glycan core al,6-
linked fucose and (31,4-linked bisecting G1cNAc, group II preferably
corresponds to bisecting
G1cNAc type N-glycans while group I preferentially corresponds to other
terminal HexNAc
containing N-glycans, preferentially with a branching HexNAc in the N-glycan
core structure, more
preferentially including structures with a branching G1cNAc in the N-glycan
core structure. In a
specific embodiment the glycan structures of this group includes core
fucosylated bisecting G1cNAc
comprising N-glycan, wherein the additional G1cNAc is G1cNAc(341inked to
Man(34G1cNAc
epitope forming epitope structure G1cNAc(34Man(34GlcNAc preferably between the
complex type
N-glycan branches.
In a preferred embodiment of the present invention, such structures include
G1cNAc linked to the 2-
position of the (31,4-linked mannose. In a further preferred embodiment of the
present invention,
such structures include G1cNAc linked to the 2-position of the (31,4-linked
mannose as described for
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LEC14 structure (Raju and Stanley J. Biol Chem (1996) 271, 7484-93), this is
specifically preferred
embodiment, supported by analysis of gene expression data and
glycosyltransferase specificities. In
a further preferred embodiment of the present invention, such structures
include G1cNAc linked to
the 6-position of the (31,4-linked G1cNAc of the N-glycan core as described
for LEC 14 structure
(Raju, Ray and Stanley J. Biol Chem (1995) 270, 30294-302).
The invention is specifically directed to further analysis of the subtypes of
the group I glycans
comprising structures according to the group I. The invention is further
directed to production of
specific binding reagents against the N-glycan core marker structures and use
of these for analysis
of the preferred cancer marker structures. The invention is further directed
to the analysis of LEC 14
and/or 18 structures by negative recognition by lectins PSA (pisum sativum) or
Intil (Lens culinaris)
lectin or core Fuc specific monoclonal antibodies, which binding is prevented
by the GIcNAcs.
Invention is specifically directed to N-glycan core marker structure, wherein
the disaccharide
epitope is Man(34G1cNAc structure in the core structure of N-linked glycan
according to the
Formula CGN:
[Mana3]ni(Mana6) n2Man(34G1cNAc(34(Fuca6)n3GlcNAcxR,
wherein nl, n2 and n3 are integers 0 or 1, independently indicating the
presence or
absence of the residues, and
wherein the non-reducing end terminal Mana3/Mana6- residues can be elongated
to the
complex type, especially biantennary structures or to mannose type (high-Man
and/or low Man)
or to hybrid type structures for the analysis of the status of stem cells
and/or manipulation of the
stem cells, wherein xR indicates reducing end structure of N-glycan linked to
protein or petide
such as (3Asn or (3Asn-peptide or (3Asn-protein, or free reducing end of N-
glycan or chemical
derivative of the reducing produced for analysis.
The invention is further directed to the N-glycan core marker structure and
marker glycan
compositions comprising structures of Formula CGN, wherein Mana3/Mana6-
residues are
elongated to the complex type, especially biantennary structures and n3 is 1
and wherein the
Man(34GlcNAc-epitope comprises the G1cNAc substitutions.
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The invention is further directed to the N-glycan core marker structure and
marker glycan
compositions comprising structures of Formula CGN, wherein Mana3/Mana6-
residues are
elongated to the complex type, especially biantennary structures and 0 is 1
and wherein the Man(34G1cNAc-epitope comprises between 1-8 % of the G1cNAc
substitutions.
The invention is further directed to the N-glycan core marker structure and
marker glycan
compositions comprising structures of Formula CGN, wherein the structure is
selected from the
group:
[G1cNAc(32Mana3](G1cNAc(32Man(x6) Man(34G1cNAc(34(Fuc(x6)n3G1cNAcxR,
[Gal(34G1cNAc(32Mana3](Gal(34G1eNAc(32Mana6) Man(34G1cNAc(34(Fuca6)n3G1cNAcxR,
and sialylated variants thereof when SA is 0 and or a6-linked to one or two
Gal residues and
Man(34 or G1cNAc(34 is substituted by G1cNAc.
The invention is further directed to the N-glycan core marker structure and
marker glycan
compositions comprising of Formula CGN, wherein the Man(34G1cNAc-epitope
comprises and the
G1cNAc residue is (32-linked to Man(34 forming epitope G1cNAc(32Man(34.
The invention is further directed to the N-glycan core marker structure and
marker glycan
compositions comprising of Formula CGN, wherein the Man(34G1cNAc-epitope
comprises and the
G1cNAc residue is 6-linked to G1cNAc of the epitope forming epitope
Man(34(G1cNAc6)G1cNAc.
The invention is further directed to the N-glycan core marker structure and
marker glycan
compositions comprising of Formula CGN, wherein the Man(34G1cNAc-epitope
comprises and the
G1cNAc residue is 4-linked to G1cNAc of the epitope forming epitope
G1cNAc(34Man(34G1cNAc.
Analysis of specific glycan groups in hESC glycomes
The analysis of N-glycome revealed signals and monosaccharide compositions
specific for
embryonic stem cells at various differentiation levels. Some preferred
structures are assigned in
Tables 12 and 13. The terminal structures were assigned based on specific
binding molecules NMR
and glycosidase digestions. The binding molecules for terminal epitopes
including structures
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present also in glycolipids or on proteins and lipids are indicated in Tables
14-19. The invention is
directed to specific reagents recognizing the preferred terminal epitopes on N-
glycans.
Over view of 50 most common structures
Neutral 2lycans
Figure 7 shows neutral glycans at three differentiation stages. The structures
of glycans are
indicated by symbols based on the recommendations of Consortium for Functional
Glycomics. The
glycans include terminal mannose comprising structures with regular high-
mannose structures and
low mannose structures, with characteristic changes during differentiation.
The mannose glycans further includes single HexNAc comprising structures
H4_ioNi, which also
change during differentiation. A specifically characteric glycans have
compositions H4N1 and
H5Nl,which increase during differentiation from stage 1(ES cells) to stage 2
(EB) and further to
stage 3. The other signal in this group (H6N1, H7N1, H8N1, H9N1 and HlONl
increase to stage 2
but the decrease.
The glycans are assigned as degradation products of High/Low mannose or even
hybrid type
structures. A preferred structural assignment is directed to glycans with
High/Low mannose
structures comprising single GIcNAc unit at the reducing end. This type of
glycans have been
known from free cytosolic glycans as degradation products of N-glycans. The
glycans are produced
by endo-beta-N-acetylglucosaminidase (chitobiosidase) cleaving the glycan
between the G1cNAc
residues. It is realized that the glycan pool may also comprise hybrid type
glycans released by endo-
beta-mannosidase. The product would comprise N-acetyllactosamine on one branch
and mannose
residues on the other branch (lower variant of H4N1).
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A selection of hybrid and complex type glycans are showns in Figure 8. The
glycans includes
hybrid type (and(or monoantennary glycans). In this first group (left) signal
H3N3 shows major
change from stage 2 to stage 3, and H2N4F1 from stage 1 to stage 3. The
glycans classified as
complex type structures in the middle also change during differentiation. The
major signals
corresponding to biantennary N glycans H5N4 and H5N4F1 decrease during the
differentiation
similarily as difucosylated structure H5N4F2 and multilactosaminylated H6N5
and H6N5F1
structures preferably corresponding to triantennary glycans. The structures
increasing during the
differentiation includes H4N4, H3N5F1, H4N5F3, and H5N5 (structural scheme is
lacking terminal
Gal or hexose units).
Acidic glycans
The figure 9 indicates 50 most abundant acidic glycans. The major complex type
N-glycan signals
with sialic acids S1H5N4F1 and S1H5N4F2 decrease during differentiation, while
the amounts of
sulfated structures H5N4F 1 P, and S 1 H5N4F 1 P (P indicates sulfate or
fosfate, ) similarily as a
structure comprising additional HexNAc (S1H5N5F1) increases.
The figure 10 shows approximated relative amounts of hydrid type glycans
indicating quite similar
amounts of acidic and neutral hydrid/monoantenanry glycans. The relative
amounts of both glycan
types increases during differentiation. Sulfated (or fosforylated) glycans are
increased among the
hybrid type glycans.
The glycans changing during differentiation with composition S1H6N4F1Ac,
S1H6N4F2, and
H6N4 in a specific embodiment include biantennary structures with additional
terminal hexose,
which may be derived from exogenous proteins, in a specific embodiment the
hexose is Gala3-
structure.
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Figures 11 and 12 includes high and Low mannose structures. The changes of the
low mannose
structures during the differentiation are characteristic for the stem cells.
The smallest low mannose
structure (H1N2) decreases while larger ones increase.
Neutral and acidic fucosylated glycans are presented in Fig. 13 Among the
entral fucosylated
glycans the amounts of apparently degraded low mannose group structures are
increased (H2N2F 1,
H3N2F1 and H3N3F1), while the complex type structures decrease similarily in
acidic and neutral
glycans except the structure with additional HexNAe, S1H5N5F1.
Figure 14 shows the neutral and acidic glycans comprising at least two fucose
residues. These are
considered as comprising fucosylated lactosamine and referred as
complex/complexly fucosylated
structures. In general decrease of the complexly fucosylated structures is
observed except the
structures with additional HexNAc residues, H4N4F2 (potential degradation
product), H5N5F3,
H5N6F3.
Preferred sulfated marker structures in N-glycome of embzyonic stem cells
Figure 15 represents sulfated N-glycans of human embryonic stem cells and
changes in their relative
abundance during differentiation. There is major changes during
differentiation. The invention is
directed to use of the signals, monosaccharide compositions and structures
indicated as increasing
in Figure 15 for markers of differentiating embryonic stem cells. Experiments
by cleavage by
specific fosfatase enzyme and high resolution mass spectrometry indicate that
the structures with
complex type N-glycans with N-acetyllactosamine residues preferably carry
sulfate residues (sulfate
ester structures) and the Mannose type N-glycans such as high Mannose N-
glycans preferably
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carries fosfate residue(s). It is realised that the sulphated and/or
fosforylated glycomes from stem
cells are new inventive markers.
The invention is especially directed to the recognition of sulphated N-
acetyllactosamines as
differentiation markers of stem cells, embryonic stem cells. The invention is
directed to testing and
selectin optimal stem cell recognizing binder molecule, preferably antibodies
such as monoclonal
antibodies, recognizing preferred sulphated lactosamines including type
I(Gal(33G1cNAc) and type
II lactosamines (Gal04G1cNAc) comprising sulfate residue(ester) at either
position 3 or 6 of Gal
and/or on position 6 of G1cNAc. The invention is especially directed to the
recognition of the
sulphated lactosamines from an N-glycan composition as shown by the invention.
Large N-glycan structure
Figure 16. shows large N-glycans (H>7, N>6) of human embryonic stem cells and
changes in their
relative abundance during differentiation. Figure 16 represents large N-
glycans of human embryonic
stem cells and changes in their relative abundance during differentiation.
There is major changes
during differentiation. The invention is directed to use of the signals,
monosaccharide compositions
and structures indicated as increasing in Figure 16 for markers of
differentiating embryonic stem
cells.
The invention reveals that the N-glycans of embryonic stem cells comprise
multiantennary N-
aglycans with at least three antennae with characteristic differntiation
associated cahges. The
invention reveals even much larger N-glycans containin poly-N-acetyllctosamine
glycans. The
invention is especially directed to use of reagents recognizing linear
(example of preferred regent
potato lectin, Solanum tuberosum agglutinin, STA) or branced poly-N-
acetyllactosamine. The
results revealed that recognition of branched N-acetyllactosamines is
especially useful for
characterization or separation or manipulation of embyronal stem cells.
Preferred reagents includes
PWA, pokeweed agglutinin and/or antibody recognizing brancehed poly-N-
acetyllactosamines such
as I-blood group antibodies.
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Cell types
In the present text, cell types refer to stem cells, especially human
embryonic stem cells (hESC) and
cells differentiated from them, preferentially embryoid bodies (EB) and stage
3 (st.3) and further
differentiated cells.
Glycan dataset and glycan profile analysis
The present invention is directed to analysing glycan profiles to enable uses
including the
following:
1. comparison between stem cell and differentiated samples,
2. comparison between different samples of the same cell type,
3. identification of differentiation stage,
4. identification of glycan signals and glycan structures associated with
different cell types or
differentiation stages,
5. identification of glycan signal groups and glycan structure groups
associated with different
cell types or differentiation stages,
6. identification of biosynthetic glycan groups associated with different cell
types or
differentiation stages,
7. identification of glycan fingerprints and glycan signatures, i.e. glycan
profiles or subprofiles
therefrom, respectively, which are associated with different cell types or
differentiation
stages, and
8. evaluating glycans or glycan groups with respect to their degree of
association with given
cell type.
As described in the present invention, analysis of multiple samples from the
same cell type reveals
that some glycans or glycan groups are constantly associated with given cell
type, whereas other
glycans or glycan groups vary individually or between different samples within
the same cell type.
The present invention is especially directed to analyzing multiple samples of
a given cell type to
reach a point of statistical confidence, preferentially over 95% confidence
level and even more
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preferentially over 96% confidence level, where given cell type or the glycan
types associated with
it can be reliably identified.
The present invention is specifically directed to comparison of multiple
glycan profile data to find
out which glycan signals are consistently associated with given cell type or
not present in it, which
are constant in all cell types, which are subject to individual or cell line
specific variation, and
which are indicative for the absence or presence of certain differentiation
stages or lineages, more
preferentially pluripotency (stem cell) or neuroectodermal differentation. The
inventors found that
the N-glycan profiles of human embryonic stem cells and cell derived from them
contain glycan
signals and glycan signal groups with the properties described above.
The present invention is further directed to establishing reference datasets
from single glycan
signals or glycan fingerprints or signatures (profiles or subprofiles), which
can be reliably used for
quality control, estimation of differential properties of new samples, control
of variation between
samples, or estimation of the effects of external factors or culture
conditions on cell status. In this
aspect of the invention, data acquired from new sample are compared to
reference dataset with a
predetermined equation to evaluate the status of the sample.
Structure specific glycan binding reagents
The present invention is further directed to using knowledge of glycan
features associated with
different cell types or differentiation stages to design glycan-binding
reagents, more preferably
glycan-binding proteins, for specific identification of stem cells or
differentiated cells. The present
invention is further directed to using such structure specific reagents to
specifically recognize, label,
or tag either specific stem cell or specific differentiated cell types, more
preferentially animal feeder
cells and more preferably mouse feeder cells. Such labels or tags can then be
used to isolate and/or
remove such cells by methods known in the art.
The binding methods for recognition of structures from cell surfaces
Recognition of structures from glycome materials and on cell surfaces by
binding methods
The present invention revealed that beside the physicochemical analysis by NMR
and/or mass
spectrometry several methods are useful for the analysis of the structures.
The invention is
especially directed to two methods:
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i) Recognition by enzymes involvingbinding and alteration of structures.
This method alters specific glycan structures by enzymes cabable of altering
the glycan
structures. The preferred enzymes includes
a) glycosidase-type enzymes capable of releasing monosaccharide units from
glycans
b) glycosyltransferring enzymes, including transglycosylating enzymes and
glycosyltransferases
c) glycan modifying enzymes including sulfate and or fosfate modifying enzymes
ii) Recognition by molecules binding glycans referred as the binders
These molecules bind glycans and include property allowing observation of the
binding such as
a label linked to the binder. The preferred binders include
a) Proteins such as antibodies, lectins and enzymes
b) Peptides such as binding domains and sites of proteins, and synthetic
library derived
analogs such as phage display peptides
c) Other polymers or organic scaffold molecules mimicking the peptide
materials
The peptides and proteins are preferably recombinant proteins or corresponding
carbohydrate
recognition domains derived therereof, when the proteins are selected from the
group monoclonal
antibody, glycosidase, glycosyl transferring enzyme, plant lectin, animal
lectin or a peptide mimetic
thereof, and wherein the binder includes a detectable label structure..
Preferred binder molecules
The present invention revealed various types of binder molecules useful for
characterization of cells
according to the invention and more specifically the preferred cell groups and
cell types according
to the invention. The preferred binder molecules are classified based on the
binding specificity with
regard to specific structures or structural features on carbohydrates of cell
surface. The preferred
binders recognize specifically more than single monosaccharide residue.
It is realized that most of the current binder molecules such as all or most
of the plant lectins are not
optimal in their specificity and usually recognize roughly one or several
monosaccharides with
various linkages. Furthermore the specificities of the lectins are usually not
well characterized with
several glycans of human types.
The preferred high specificity binders recognize
A) at least one monosaccharide residue and a specific bond structure between
those to another
monosaccharides next monosaccharide residue referred as MS1B1-binder,
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B) more preferably recognizing at least part of the second monosaccharide
residue referred as
MS2B1-binder,
C) even more preferably recognizing second bond structure and or at least part
of third mono
saccharide residue, referred as MS3B2-binder, preferably the MS3B2 recognizes
a specific
complete trisaccharide structure.
D) most preferably the binding structure recognizes at least partially a
tetrasaccharide with
three bond structures, referred as MS4B3-binder, preferably the binder
recognizes complete
tetrasaccharide sequences.
The preferred binders includes natural human and or animal, or other proteins
developed for
specific recognition of glycans. The preferred high specificity binder
proteins are specific
antibodies preferably monoclonal antibodies; lectins, preferably mammalian or
animal lectins; or
specific glycosyltransferring enzymes more preferably glycosidase type
enzymes,
glycosyltransferases or transglycosylating enzymes.
Target structures for specific binders and examples of the binding molecules
Combination of terminal structures in combination with specific zlycan core
structures
It is realized that part of the structural elements are specifically
associated with specific glycan core
structure. The recognition of terminal structures linked to specific core
structures are especially
preferred, such high specificity reagents have capacity of recognition almost
complete individual
glycans to the level of physicochemical characterization according to the
invention. For example
many specific mannose structures according to the invention are in general
quite characteristic for
N-glycan glycomes according to the invention. The present invention is
especially directed to
recognition terminal epitopes.
Common terminal structures on several zlycan core structures
The present invention revealed that there are certain common structural
features on several glycan
types and that it is possible to recognize certain common epitopes on
different glycan structures by
specific reagents when specificity of the reagent is limited to the terminal
without specificity for the
core structure. The invention especially revealed characteristic terminal
features for specific cell
types according to the invention. The invention realized that the common
epitopes increase the
effect of the recognition. The common terminal structures are especially
useful for recognition in
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the context with possible other cell types or material, which do not contain
the common terminal
structure in substancial amount.
Specific preferred structural groups
The present invention is directed to recognition of oligosaccharide sequences
comprising specific
terminal monosaccharide types, optionally further including a specific core
structure. The preferred
oligosaccharide sequences classified based on the terminal monosaccharide
structures.
1. Structures with terminal Mannose monosaccharide
Preferred mannose-type target structures have been specifically classified by
the invention. These
include various types of high and low-mannose structures and hybrid type
structures according to
the invention.
Low or uncharacterised speci acity binders
preferred for recognition of terminal mannose structures includes mannose-
monosaccharide binding
plant lectins.
Preferred high specific high specificity binders
include
i) Specific mannose residue releasing enzymes such as linkage specific
mannosidases, more
preferably an a-mannosidase or (3-mannosidase.
Preferred a-mannosidases includes linkage specific (x-mannosidases such as a-
Mannosidases
cleaving preferably non-reducing end terminal
a2-linked mannose residues specifically or more effectively than other
linkages, more preferably
cleaving specifically Mana2-structures; or
a6-linked mannose residues specifically or more effectively than other
linkages, more preferably
cleaving specifically Mana6-structures;
Preferred (3-mannosidases includes (3-mannosidases capable of cleaving 04-
linked mannose from
non-reducing end terminal of N-glycan core Man(34G1cNAc-structure without
cleaving other 0-
linked monosaccharides in the glycomes.
ii) Specific binding proteins recognizing preferred mannose structures
according to the invention.
The preferred reagents include antibodies and binding domains of antibodies
(Fab-fragments and
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like), and other engineered carbohydrate binding proteins. The invention is
directed to antibodies
recognizing MS2B1 and more preferably MS3B2-structures
2. Structures with terminal Gal- monosaccharide
Preferred galactose-type target structures have been specifically classified
by the invention. These
include various types of N-acetyllactosamine structures according to the
invention.
Low or uncharacterised sPeci acity binders for terminal Gal
Prereferred for recognition of terminal galactose structures includes plant
lectins such as ricin lectin
(ricinus communis agglutinin RCA), and peanut lectin(/agglutinin PNA).
Preferred high specific high specifcity binders include
i) Specific galactose residue releasing enzymes such as linkage specific
galactosidases, more
preferably a-galactosidase or(3-galactosidase.
Preferred a-galactosidases include linkage galactosidases capable of cleaving
Gala3Gal-structures
revealed from specific cell preparations
Preferred (3-galactosidases includes (3- galactosidases capable of cleaving
(34-linked galactose from non-reducing end terminal Gal(34GlcNAc-structure
without cleaving other
(3-linked monosaccharides in the glycomes and
(33-linked galactose from non-reducing end terminal Gal(33GlcNAc-structure
without cleaving other
(3-linked monosaccharides in the glycomes
ii)Specific binding proteins recognizing preferred galactose structures
according to the invention.
The preferred reagents include antibodies and binding domains of antibodies
(Fab-fragments and
like), and other engineered carbohydrate binding proteins and animal lectins
such as galectins.
3. Structures with terminal GaINAc- monosaccharide
Preferred GaLNAc-type target structures have been specifically revealed by the
invention. These
include especially LacdiNAc, Ga1NAc(3G1cNAc-type structures according to the
invention.
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Low or uncharacterised specifcity binders for terminal GaINAc
Several plant lectins has been reported for recognition of terminal Ga1NAc. It
is realized that some
GaINAc-recognizing lectins may be selected for low specificity reconition of
the preferred
LacdiNAc-structures.
Preferred hizh specific hiQh specifcity binders include
i) The invention revealed that (3-linked Ga1NAc can be recognized by specific
(3-N-
acetylhexosaminidase enzyme in combination with (3-N-acetylhexosaminidase
enzyme.
This combination indicates the terminal monosaccharide and at least part of
the linkage structure.
Preferred (3-N-acetylehexosaminidase, includes enzyme capable of cleaving (3-
linked GaINAc from
non-reducing end terminal Ga1NAc(34/3-structures without cleaving (Ainked
HexNAc in the
glycomes; preferred N-acetylglucosaminidases include enzyme capable of
cleaving (3-linked
G1cNAc but not Ga1NAc.
ii) Specific binding proteins recognizing preferred GaLNAc(34, more preferably
Ga1NAc(34G1cNAc,
structures according to the invention. The preferred reagents include
antibodies and binding
domains of antibodies (Fab-fragments and like), and other engineered
carbohydrate binding
proteins, and a special plant lectin WFA (Wisteria floribunda agglutinin).
4. Structures with terminal GIcNAc- monosacchaNide
Preferred G1cNAc-type target structures have been specifically revealed by the
invention. These
include especially G1cNAc(3-type structures according to the invention.
Low or uncharacterised specificity binders for terminal GlcNAc
Several plant lectins has been reported for recognition of terminal G1cNAc. It
is realized that some
G1cNAc-recognizing lectins may be selected for low specificity reconition of
the preferred G1cNAc-
structures.
Preferred high specific high specifcity binders include
i) The invention revealed that (3-linked G1cNAc can be recognized by specific
(3-N-
acetylglucosaminidase enzyme.
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Preferred (3-N-acetylglucosaminidase includes enzyme capable of cleaving (3-
linked G1cNAc from
non-reducing end terminal GIcNAc(32/3/6-structures without cleaving (3-linked
Ga1NAc or (x-linked
HexNAc in the glycomes;
ii) Specific binding proteins recognizing preferred G1cNAc(32/3/6, more
preferably
G1cNAc(32Man(x, structures according to the invention. The preferred reagents
include antibodies
and binding domains of antibodies (Fab-fragments and like), and other
engineered carbohydrate
binding proteins.
5. Structures with terminal Fucose- monosaccharide
Preferred fucose-type target structures have been specifically classified by
the invention. These
include various types of N-acetyllactosamine structures according to the
invention.
Low or uncharacterised specifcity binders for terminal Fuc
Prereferred for recognition of terminal fucose structures includes fucose
monosaccharide binding
plant lectins. Lectins of Ulex europeaus and Lotus tetragonolobus has been
reported to recognize
for example terminal Fucoses with some specificity binding for a2-linked
structures, and branching
a3-fucose, respectively.
Preferred hip_h speci ac hi h speci acity binders include
i) Specific fucose residue releasing enzymes such as linkage fucosidases, more
preferably a-
fucosidase.
Preferred a-fucosidases include linkage fucosidases capable of cleaving
Fuc(x2Ga1-, and
Gal(34/3(Fuca3/4)G1cNAc-structures revealed from specific cell preparations.
ii)Specific binding proteins recognizing preferred fucose structures according
to the invention. The
preferred reagents include antibodies and binding domains of antibodies (Fab-
fragments and like),
and other engineered carbohydrate binding proteins and animal lectins such as
selectins recognizing
especially Lewis type structures such as Lewis x, Gal(34(Fuca3)G1cNAc, and
sialyl-Lewis x,
SAa3 Gal(34(Fuca3)G1cNAc.
The preferred antibodies includes antibodies recognizing specifically Lewis
type structures such as
Lewis x, and sialyl-Lewis x. More preferably the Lewis x-antibody is not
classic SSEA-1 antibody,
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but the antibody recognizes specific protein linked Lewis x structures such as
Gal(34(Fuca3)G1cNAc(32Mana-linked to N-glycan core.
6. Structures with terminal Sialic acid- monosaccharide
Preferred sialic acid-type target structures have been specifically classified
by the invention.
Low or uncharacterised specifcity binders for terminal Fuc
Preferred for recognition of terminal sialic acid structures includes sialic
acid monosaccharide
binding plant lectins.
Preferred hip_h specific hi h sPeci acity binders include
i) Specific sialic acid residue releasing enzymes such as linkage sialidases,
more preferably a-
sialidases.
Preferred a-sialidases include linkage sialidases capable of cleaving SAa3Gal-
and SAa6Ga1-
structures revealed from specific cell preparations by the invention.
Preferred lectins, with linkage specificity include the lectins, that are
specific for SAa3Ga1-
structures, preferably being Maackia amurensis lectin and/or lectins specific
for SAa6Ga1-
structures, preferably being Sambucus nigra agglutinin.
ii)Specific binding proteins recognizing preferred sialic acid oligosaccharide
sequence structures
according to the invention. The preferred reagents include antibodies and
binding domains of
antibodies (Fab-fragments and like), and other engineered carbohydrate binding
proteins and animal
lectins such as selectins recognizing especially Lewis type structures such as
sialyl-Lewis x,
SAa3Gal(34(Fuca3)G1cNAc or sialic acid recognizing Siglec-proteins.
The preferred antibodies includes antibodies recognizing specifically sialyl-N-
acetyllactosamines,
and sialyl-Lewis x.
Preferred antibodies for NeuGc-structures includes antibodies recognizes a
structure
NeuGca3Gal(34G1c(NAc)o or i and/or Ga1NAc(34[NeuGca3]Gal(34G1c(NAc)o or i,
wherein []
indicates branch in the structure and Oo or 1 a structure being either present
or absent. In a preferred
embodiment the invention is directed recognition of the N-glycolyl-Neuraminic
acid structures by
antibody, preferably by a monoclonal antibody or human/humanized monoclonal
antibody. A
preferred antibody contains the variable domains of P3-antibody.
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Binder-label conjuizates
The present invention is specifically directed to the binding of the
structures according to the
present invention, when the binder is conjugated with "a label structure". The
label structure means
a molecule observable in a assay such as for example a fluorescent molecule, a
radioactive
molecule, a detectable enzyme such as horse radish peroxidase or
biotin/streptavidin/avidin. When
the labelled binding molecule is contacted with the cells according to the
invention, the cells can be
monitored, observed and/or sorted based on the presence of the label on the
cell surface. Monitoring
and observation may occur by regular methods for observing labels such as
fluorescence measuring
devices, microscopes, scintillation counters and other devices for measuring
radioactivity.
Use of binder and labelled binder-conjugates for cell sorting
The invention is specifically directed to use of the binders and their
labelled cojugates for sorting or
selecting cells from biological materials or samples including cell materials
comprising other cell
types. The preferred cell types includes cultivated cells and associated cells
such as feeder cells.
The labels can be used for sorting cell types according to invention from
other similar cells. In
another embodiment the cells are sorted from different cell types such as
blood cells or in context of
cultured cells preferably feeder cells, for example in context of complex cell
cultures
corresponding feeder cells such as human or mouse feeder cells. A preferred
cell sorting method is
FACS sorting. Another sorting methods utilized immobilized binder structures
and removal of
unbound cells for separation of bound and unbound cells.
Use of immobilized binder structures
In a preferred embodiment the binder structure is conjugated to a solid phase.
The cells are
contacted with the solid phase, and part of the material is bound to surface.
This method may be
used to separation of cells and analysis of cell surface structures, or study
cell biological changes of
cells due to immobilization. In the analytics involving method the cells are
preferably tagged with
or labelled with a reagent for the detection of the cells bound to the solid
phase through a binder
structure on the solid phase. The methods preferably further include one or
more steps of washing
to remove unbound cells.
Preferred solid phases include cell suitable plastic materials used in
contacting cells such as cell
cultivation bottles, petri dishes and microtiter wells; fermentor surface
materials
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Specific recognition between preferred stem cells and contaminating cells
The invention is further directed to methods of recognizing stem cells from
differentiated cells such
as feeder cells, preferably animal feeder cells and more preferably mouse
feeder cells. It is further
realized, that the present reagents can be used for purification of stem cells
by any fractionation
method using the specific binding reagents.
Preferred fractionation methods includes fluorecense activated cell sorting
(FACS), affinity
chromatography methods, and bead methods such as magnetic bead methods.
Preferred reagents for recognition between preferred cells, preferably
embryonic type cells, and
and contaminating cells, such as feeder cells most preferably mouse feeder
cells, includes reagents
according to the Table 43, more preferably proteins with similar specificity
with lectins PSA, MAA,
and PNA.
The invention is further directed to positive selection methods including
specific binding to the stem
cell population but not to contaminating cell population. The invention is
further directed to
negative selection methods including specific binding to the contaminating
cell population but not
to the stem cell population. In yet another embodiment of recognition of stem
cells the stem cell
population is recognized together with a homogenous cell population such as a
feeder cell
population, preferably when separation of other materials is needed. It is
realized that a reagent for
positive selection can be selected so that it binds stem cells as in present
invention and not to the
contaminating cell population and a regent for negative selection by selecting
opposite specificity.
In case of one population of cells according to the invention is to be
selected from a novel cell
population not studied in the present invention, the binding molecules
according to the invention
maybe used when verified to have suitable specificity with regard to the novel
cell population
(binding or not binding). The invention is specifically directed to analysis
of such binding
specificity for development of a new binding or selection method according to
the invention.
The preferred specificities according to the invention includes recognition
of:
i) mannose type structures, especially alpha-Man structures like lectin PSA,
preferably on
the surface of contaminating cells
ii) 0-sialylated structures similarily as by MAA-lectin, preferably for
recognition of
embryonic type stem cells
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iii) GaUGa1NAc binding specificity, preferably Gall-3/Ga1NAc1-3 binding
specificity, more
preferably Gal(31-3/Ga1NAc(31-3 binding specificity similar to PNA, ,
preferably for
recognition of embryonic type stem cells
Manipulation of cells by binders
The invention is specifically directed to manipulation of cells by the
specific binding proteins. It is
realized that the glycans described have important roles in the interactions
between cells and thus
binders or binding molecules can be used for specific biological manipulation
of cells. The
manipulation may be performed by free or immobilized binders. In a preferred
embodiment cells
are used for manipulation of cell under cell culture conditions to affect the
growth rate of the cells.
Identification and classification of differences in glycan datasets
The present invention is specifically directed to analyzing glycan datasets
and glycan profiles for
comparison and characterization of different cell types. In one embodiment of
the invention, glycan
signals or signal groups associated with given cell type are selected from the
whole glycan datasets
or profiles and indifferent glycan signals are removed. The resulting selected
signal groups have
reduced background and less observation points, but the glycan signals most
important to the
resolving power are included in the selection. Such selected signal groups and
their patterns in
different sample types serve as a signature for the identification of the cell
type and/or glycan types
or biosynthetic groups that are typical to it. By evaluating multiple samples
from the same cell type,
glycan signals that have individual i.e. cell line specific variation can be
excluded from the
selection. Moreover, glycan signals can be identified that do not differ
between cell types, including
major glycans that can be considered as housekeeping glycans.
To systematically analyze the data and to find the major glycan signals
associated with given cell
type according to the invention, difference-indicating variables can be
calculated for the comparison
of glycan signals in the glycan datasets. Preferential variables between two
samples include
variables for absolute and relative difference of given glycan signal between
the datasets from two
cell types. Most preferential variables according to the invention are:
1. absolute difference A = (S2 - SI ), and
2. relative difference R = A / SI,
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wherein SI and S2 are relative abundances of a given glycan signal in cell
types I and 2,
respectively.
It is realized that other mathematical solutions exist to express the idea of
absolute and relative
difference between glycan datasets, and the above equations do not limit the
scope of the present
invention. According to the present invention, after A and R are calculated
for the glycan profile
datasets of the two cell types, the glycan signals are thereafter sorted
according to the values of A
and R to identify the most significant differing glycan signals. High value of
A or R indicates
association with cell type 2, and vice versa. In the list of glycan data
sorted independently by R and
A, the cell-type specific glycans occur at the top and the bottom of the
lists. More preferentially, if a
given signal has high values of both A and R, it is more significant.
Preferred representation of the dataset when comparing two cell materials
The present invention is specifically directed to the comparative presentation
of the quantitative
glycome dataset as multidimensional graphs comparing the paraller data for
example as shown in
figures or as other three dimensional presentations as for example as two
dimensional matrix
showing the quantities with a quantitative code, preferably by a quantitative
color code.
Released glycomes
The invention is directed to methods to produce released, in a preferred
enzymatically released
glycans, also referred as glycomes, from embryonic type cells. A preferred
glycome type is N-
glycan glycome released by a N-glycosidase enzyme. The invention is further
directed to profiling
analysis of the released glycomes.
Low amounts of cells for glycome analysis from stem cells
The invention revealed that its possible to produce glycome from very low
amount of cells. The
preferred embodiments amount of cells is between 1000 and 10 000 000 cells,
more preferably
between 10 000 and 1 000 000 cells. The invention is further directed to
analysis of released
glycomes of amount of at least 0.1 pmol, more preferably of at least to 1
pmol, more preferably at
least of 10 pmol.
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(a) Total asparagine-linked glycan (N-glycan) pool was enzymatically isolated
from about 100 000
cells. (b) The total N-glycan pool (picomole quantities) was purified with
microscale solid-phase
extraction and divided into neutral and sialylated N-glycan fractions. The N-
glycan fractions were
analyzed by MALDI-TOF mass spectrometry either in positive ion mode for
neutral N-glycans (c)
or in negative ion mode for sialylated glycans (d). Over one hundred N-glycan
signals were
detected from each cell type revealing the surprising complexity of hESC
glycosylation. The
relative abundances of the observed glycan signals were determined based on
relative signal
intensities (Saarinen et al., 1999, Eur. J. Biochem. 259, 829-840).
Preferred structures of 0-glycan glycomes of stem cells
The present invention is especially directed to following 0-glycan marker
structures of stem cells:
Core 1 type 0-glycan structures following the marker composition
NeuAc2Hex,HexNAci,
preferably including structures SAa3Gal(33Ga1NAc and/or
SAa3Ga1(33(Saa6)GaLNAc;
and Core 2 type 0-glycan structures following the marker composition NeuAco_
zHexzHexNAczdHexo_i, more preferentially further including the glycan series
NeuAco_
zHexz+nHexNAc2+ndHexo_i, wherein n is either 1, 2, or 3 and more
preferentially n is 1 or 2, and
even more preferentially n is 1;
more specifically preferably including RiGal(34(R3)GIcNAc(36(RzGal(33)Ga1NAc,
wherein Ri and R2 are independently either nothing or sialic acid residue,
preferably a2,3-linked
sialic acid residue, or an elongation with HexHexNAcn, wherein n is
independently an integer at
least 1, preferably between 1-3, most preferably between 1-2, and most
preferably 1, and the
elongation may terminate in sialic acid residue, preferably a2,3-linked sialic
acid residue; and
R3 is independently either nothing or fucose residue, preferably a1,3-linked
fucose residue.
It is realized that these structures correlate with expression of P6G1cNAc-
transferases synthesizing
core 2 structures.
Preferred branched N-acetyllactosamine type glycosphingolipids
The invention furhter revealed branched, I-type, poly-N-acetyllactosamines
with two terminal
Gal(34-residues from glycolipids of human stem cells. The structures correlate
with expression of
(36G1cNAc-transferases capable of branching poly-N-acetyllactosamines and
further to binding of
lectins specific for branched poly-N-acetylalctosamines. It was further
noticed that PWA-lectin had
an activity in manipulation of stem cells, especially the growth rate thereof.
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Analysis and utilization of poly-N-acetyllactosamine sequences and non-
reducing terminal
epitopes associated with different glycan types
The present invention is directed to poly-N-acetyllactosamine sequences (poly-
LacNAc) associated
with cell types accoriding to the present invention. The inventors found that
different types of poly-
LacNAc are characteristic to different cell types, as described in the
Examples of the present
invention. hESC are characterized by type 1 terminating poly-LacNAc,
especially on 0-glycans and
glycolipids. The present invention is especially directed to the analysis and
utilization of these
glycan characteristics according to the present invention. The present
invention is further directed to
the analysis and utilization of the specific cell-type accociated glycan
sequences revealed in the
present Examples according to the present invention.
The present invention is directed to non-reducing terminal epitopes in
different glycan classes
including N- and 0-glycans, glycosphingolipid glycans, and poly-LacNAc. The
inventors found
that especially the relative amounts of (31,4-linked Gal, (31,3-linked Gal,
al,2-linked Fuc, al,3/4-
linked Fuc, a-linked sialic acid, and a2,3-linked sialic acid are
characteristically different between
the studied cell types; and the invention is especially directed to the
analysis and utilization of these
glycan characteristics according to the present invention.
The present invention is further directed to analyzing fucosylation degree in
0-glycans by
comparing indicative glycan signals such as neutral 0-glycan signals at mlz
771 and 917 as
described in the Examples. The inventors found that compared to other cell
types analyzed in the
present invention, hESC had low relative abundance of neutral 0-glycan signal
at m/z 917
compared to 771, indicating low fucosylation degree of the 0-glycan sequences
corresponding to
the signal at m/z 771 and containing terminal (31,4-linked Gal. Another
difference was the
occurrence of abundant signal at m/z 552 in hESC, corresponding to
HexiHexNAcidHexi,
including a1,2-fucosylated Core 1 0-glycan sequence. In contrast, in CB MNC
the glycan signal at
m/z 917 is relatively abundant, indicating high fucosylation degree of the O-
glycan sequences
corresponding to the signal at m/z 771 and containing terminal (31,4-linked
Gal. The other cell types
analyzed in the present invention also had characteristic fucosylation degree
between these two cell
types.
Especially, the present invention is directed to analyzing terminal epitopes
associated with poly-
LacNAc in stem cells, more preferably when these epitopes are presented in the
context of a poly-
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LacNAc chain, most preferably in 0-glycans or glycosphingolipids. The present
invention is further
directed to analyzing such characteristic poly-LacNAc, terminal epitope, and
fucosylation profiles
according to the methods of the present invention, in glycan structural
characterization and specific
glycosylation type identification, and other uses of the present invention;
especially when this
analysis is done based on endo-(3-galactosidase digestion, by studying the non-
reducing terminal
fragments and their profile, and/or by studying the reducing terminal
fragments and their profile, as
described in the Examples of the present invention. The inventors found that
cell-type specific
glycosylation features are efficiently reflected in the endo-(3-galactosidase
reaction products and
their profiles. The present invention is further directed to such reaction
product profiles and their
analysis according to the present invention.
Especially in hESC, the inventors found that characteristic non-reducing poly-
LacNAc associated
sequences include Fuca2Gal, Gal(33G1cNAc, Fuca2Gal(33GlcNAc, and a3'-
sialylated
Gal(33GlcNAc. The present invention is especially directed to analysis of such
glycan structures
according to the present methods, in context of stem cells and differentiation
of stem cells,
preferably in context of human embryonic stem cells and their differentiation.
The inventors further found that all three most thoroughly analyzed cellular
glycan classes, N-
glycans, 0-glycans, and glycosphingolipid glycans, were differently regulated
compared to each
other, especially with regard to non-reducing terminal glycan epitopes and
poly-LacNAc sequences
as described in the Examples and Tables of the present invention. Therefore,
combining quantitative
glycan profile analysis data from more than one glycan class will yield
significantly more
information. The present invention is especially directed to combining glycan
data obtained by the
methods of the present invention, from more than one glycan class selected
from the group of N-
glycans, 0-glycans, and glycosphingolipid glycans; more preferably, all three
classes are analyzed;
and use of this information according to the present invention. In a preferred
embodiment, N-glycan
data is combined with 0-glycan data; and in a further preferred embodiment, N-
glycan data is
combined with glycosphingolipid glycan data.
Lactosamines Ga1(33/4G1cNAc and glycolipid structures comprising lactose
structures
(Gal(34G1c)
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The lactosamines form a preferred structure group with lactose-based
glycolipids. The structures
share similar features as products of 03/4Gal-transferases. The 03/4 galactose
based structures were
observed to produce characteristic features of protein linked and glycolipid
glycomes.
The invention revealed that furthermore Ga1(33/4G1cNAc-structures are a key
feature of
differentiation releated structures on glycolipids of various stem cell types.
Such glycolipids
comprise two preferred structural epitopes according to the invention. The
most preferred glycolipid
types include thus lactosylceramide based glycosphingolipids and especially
lacto- (Gal(33G1cNAc),
such as
lactotetraosylceramide Gal(33GlcNAc(33Gal(34G1c(3Cer, prefered structures
further including its
non-reducing terminal structures selected from the group: Gal(33(Fuca4)G1cNAc
(Lewis a),
Fuca2Gal(33G1cNAc (H-type 1), structure and, Fuca2Gal(33(Fuca4)G1cNAc (Lewis
b) or sialylated
structure SAa3Ga1(33G1cNAc or SAa3Gal(33(Fuca4)G1cNAc, wherein SA is a sialic
acid,
preferably Neu5Ac preferably replacing Ga1(33G1cNAc of lactotetraosylceramide
and its fucosylated and/or elogated variants such as preferably
according to the Formula:
(Saca3)ns(Fuca2)niGal(33(Fuca4)n3GlcNAc(33
[Gal(33/4(Fuca4/3)nzGlcNAc(33]n4Ga1(34G1c(3Cer
wherein
nl is 0 or 1, indicating presence or absence of Fuca2;
n2 is 0 or 1, indicating the presence or absence of Fuca4/3 (branch),
n3 is 0 or 1, indicating the presence or absence of Fuca4 (branch)
n4 is 0 or 1, indicating the presence or absence of (fucosylated) N-
acetyllactosamine elongation;
n5 is 0 or 1, indicating the presence or absence of Saca3 elongation;
Sac is terminal structure, preferably sialic acid, with 0- linkage, with the
proviso that when Sac is
present, n5 is 1, then nl is 0
and
neolacto (Gal(34G1cNAc)-comprising glycolipids such as
neolactotetraosylceramide Gal(34G1cNAc(33Ga1(34Glc(3Cer, preferred structures
further including its
non-reducing terminal Gal(34(Fuca3)G1cNAc (Lewis x), Fuca2Gal(34G1cNAc H-type
2, structure
and, Fuca2Gal(34(Fuca3)G1cNAc (Lewis y)
and
its fucosylated and/or elogated variants such as preferably
(Saca3/6)ns(Fuc(y2)niGal(34(Fuc(x3)n3G1cNAc(33
[Gal(34(Fuc(x3)nzGlcNAc(33]n4Gal(34Glc(3Cer
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nl is 0 or 1 indicating presence or absence of Fuca2;
n2 is 0 or 1, indicating the presence or absence of Fuca3 (branch),
n3 is 0 or 1, indicating the presence or absence of Fuca3 (branch)
n4 is 0 or 1, indicating the presence or absence of (fucosylated) N-
acetyllactosamine elongation,
n5 is 0 or 1, indicating the presence or absence of Saca3/6 elongation;
Sac is terminal structure, preferably sialic acid (SA) with 0- linkage, or
sialic acid with a6-
linkage, with the proviso that when Sac is present, n5 is 1, then nl is 0, and
when sialic acid is
bound by a6- linkage preferably also n3 is 0.
Preferred stem cell lzlvcosphingolipid 2lvcan profiles, compositions, and
marker structures
The inventors were able to describe stem cell glycolipid glycomes by mass
spectrometric profiling
of liberated free glycans, revealing about 80 glycan signals from different
stem cell types. The
proposed monosaccharide compositions of the neutral glycans were composed of 2-
7 Hex, 0-5
HexNAc, and 0-4 dHex. The proposed monosaccharide compositions of the acidic
glycan signals
were composed of 0-2 NeuAc, 2-9 Hex, 0-6 HexNAc, 0-3 dHex, and/or 0-1 sulphate
or phosphate
esters. The present invention is especially directed to analysis and targeting
of such stem cell glycan
profiles and/or structures for the uses described in the present invention
with respect to stem cells.
The present invention is further specifically directed to glycosphingolipid
glycan signals specific
tostem cell types as described in the Examples. In a preferred embodiment,
glycan signals typical to
hESC, preferentially including 876 and 892 are used in their analysis, more
preferentially
FucHexHexNAcLac, wherein al,2-Fuc is preferential to al,3/4-Fuc, and
HexzHexNAc,Lac, and
more preferentially to Gal(33[HexiHexNAci]Lac.
Terminal glycan epitopes that were demonstrated in the present experiments in
stem cell
glycosphingolipid glycans are useful in recognizing stem cells or specifically
binding to the stem
cells via glycans, and other uses according to the present invention,
including terminal epitopes:
Gal, Gal(34Glc (Lac), Gal(34G1cNAc (LacNAc type 2), Ga1(33, Non-reducing
terminal HexNAc,
Fuc, a1,2-Fuc, a1,3-Fuc, Fuca2Gal, Fuca2Gal(34G1cNAc (H type 2), Fuca2Gal04G1c
(2'-
fucosyllactose), Fuca3GlcNAc, Ga1(34(Fuca3)G1cNAc (Lex), Fuca3Glc,
Gal(34(Fuca3)Glc (3-fucosyllactose), Neu5Ac, Neu5Aca2,3, and Neu5Aca2,6. The
present
invention is further directed to the total terminal epitope profiles within
the total stem cell
glycosphingolipid glycomes and/or glycomes.
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The inventors were further able to characterize in hESC the corresponding
glycan signals to SSEA-
3 and SSEA-4 developmental related antigens, as well as their molar
proportions within the stem
cell glycome. The invention is further directed to quantitative analysis of
such stem cell epitopes
within the total glycomes or subglycomes, which is useful as a more efficient
alternative with
respect to antibodies that recognize only surface antigens. In a further
embodiment, the present
invention is directed to finding and characterizing the expression of cryptic
developmental and/or
stem cell antigens within the total glycome profiles by studying total glycan
profiles, as
demonstrated in the Examples for a1,2-fucosylated antigen expression in hESC
in contrast to
SSEA-1 expression in mouse ES cells.
The present invention revealed characteristic variations (increased or
decreased expression in
comparision to similar control cell or a contaminatiog cell or like) of both
structure types in various
cell materials according to the invention. The structures were revealed with
characteristic and
varying expression in three different glycome types: N-glycans, 0-glycans, and
glycolipids. The
invention revealed that the glycan structures are a charateristic feature of
stem cells and are useful
for various analysis methods according to the invention. Amounts of these and
relative amounts of
the epitopes and/or derivatives varies between cell lines or between cells
exposed to different
conditions during growing, storage, or induction with effector molecules such
as cytokines and/or
hormones.
Preferred epitopes and antibody binders especially for analysis of embryonic
stem cells
The antibody labelling experiment Table 48 with embryonic stem cells revealed
specific of type 1
N-acetyllactosamine antigen recognizing antibodies recognizing non-modified
disaccharide
Gal(33GlcNAc (Le c, Lewis c), and fucosylated derivatives H type and Lewis
b.The antibodies were
efective in recognizing hESC cell populations in comparision to mouse feeder
cells mEF used for
cultivation of the stem cells. See Figures for results.
Specific different H type 2 recognizing antibodies were revealed to recognize
different
subpopulations of embryonic stem cells and thus usefulness for defining
subpopulations of the cells.
The invention further revealed a specific Lewis x and sialyl-Lewis x
structures on the embryonic
stem cells.
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Other preferred binders and/or antibodies comprise of binders which bind to
the same epitope than
GF 287 (H type 1). In a preferred embodiment, an antibody binds to
Fuca2Ga1(33G1cNAc epitope.
A more preferred antibody comprises of the antibody of clone 17-206 (ab3355)
by Abcam. This
epitope is suitable and can be used to detect, isolate and evaluate the
differentiation stage, and/or
plucipotency of stem cells, preferably human embryonic stem cells. The
detection can be performed
in vitro, for FACS purposes and/or for cell lineage specific purposes. This
antibody can be used to
positively isolate and/or separate and/or enrich stem cells, preferably human
embryonice stem cells
from a mixture of cells comprising feeder and stem cells.
Other preferred binders and/or antibodies comprise of binders which bind to
the same epitope than
GF 279 (Lewis c, Gal(33G1cNAc). In a preferred embodiment, an antibody binds
to
Gal(33GlcNAc epitope in glycoconjugates, more preferably in glycoproteins and
glycolipids such as
lactotetraosylceramide. A more preferred antibody comprises of the antibody of
clone K21
(ab3352) by Abcam. This epitope is suitable and can be used to detect, isolate
and evaluate the
differentiation stage, and/or plucipotency of stem cells, preferably human
embryonic stem cells.
The detection can be performed in vitro, for FACS purposes and/or for cell
lineage specific
purposes. This antibody can be used to positively isolate and/or separate
and/or enrich stem cells,
preferably human embryonice stem cells from a mixture of cells comprising
feeder and stem cells.
Other preferred binders and/or antibodies comprise of binders which bind to
the same epitope than
GF 288 (Globo H). In a preferred embodiment, an antibody binds to
Fuca2Ga1(33Ga1NAc(3 epitope,
more preferably Fuca2Ga1(33Ga1NAc(33GalaLacCer epitope. A more preferred
antibody comprises
of the antibody of clone A69-A/E8 (MAB-S206) by Glycotope. This epitope is
suitable and can be
used to detect, isolate and evaluate the differentiation stage, and/or
plucipotency of stem cells,
preferably human embryonic stem cells. The detection can be performed in
vitro, for FACS
purposes and/or for cell lineage specific purposes. This antibody can be used
to positively isolate
and/or separate and/or enrich stem cells, preferably human embryonice stem
cells from a mixture of
cells comprising feeder and stem cells.
Other preferred binders and/or antibodies comprise of binders which bind to
the same epitope than
GF 284 (H type 2). In a preferred embodiment, an antibody binds to
Fuca2Ga1(34G1cNAc epitope.
A more preferred antibody comprises of the antibody of clone B393 (DM3015) by
Acris. This
epitope is suitable and can be used to detect, isolate and evaluate the
differentiation stage, and/or
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plucipotency of stem cells, preferably human embryonic stem cells. The
detection can be performed
in vitro, for FACS purposes and/or for cell lineage specific purposes. This
antibody can be used to
positively isolate and/or separate and/or enrich stem cells, preferably human
embryonice stem cells
from a mixture of cells comprising feeder and stem cells.
Other preferred binders and/or antibodies comprise of binders which bind to
the same epitope than
GF 283 (Lewis b). In a preferred embodiment, an antibody binds to
Fuca2Ga1(33(Fuc(x4)GlcNAc
epitope. A more preferred antibody comprises of the antibody of clone 2-25LE
(DM3122) by Acris.
This epitope is suitable and can be used to detect, isolate and evaluate the
differentiation stage,
and/or plucipotency of stem cells, preferably human embryonic stem cells. The
detection can be
performed in vitro, for FACS purposes and/or for cell lineage specific
purposes. This antibody can
be used to positively isolate and/or separate and/or enrich stem cells,
preferably human embryonice
stem cells from a mixture of cells comprising feeder and stem cells.
Other preferred binders and/or antibodies comprise of binders which bind to
the same epitope than
GF 286 (H type 2). In a preferred embodiment, an antibody binds to
Fuca2Ga1(34G1cNAc epitope.
A more preferred antibody comprises of the antibody of clone B393 (BM258P) by
Acris. This
epitope is suitable and can be used to detect, isolate and evaluate the
differentiation stage, and/or
plucipotency of stem cells, preferably human embryonic stem cells. The
detection can be performed
in vitro, for FACS purposes and/or for cell lineage specific purposes. This
antibody can be used to
positively isolate and/or separate and/or enrich stem cells, preferably human
embryonice stem cells
from a mixture of cells comprising feeder and stem cells.
Other preferred binders and/or antibodies comprise of binders which bind to
the same epitope than
GF 290 (H type 2). In a preferred embodiment, an antibody binds to
Fuca2Ga1(34G1cNAc epitope.
A more preferred antibody comprises of the antibody of clone A51-B/A6 (MAB-
S204) by
Glycotope. This epitope is suitable and can be used to detect, isolate and
evaluate the differentiation
stage, and/or plucipotency of stem cells, preferably human embryonic stem
cells. The detection can
be performed in vitro, for FACS purposes and/or for cell lineage specific
purposes. This antibody
can be used to positively isolate and/or separate and/or enrich stem cells,
preferably human
embryonice stem cells from a mixture of cells comprising feeder and stem
cells.
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Other binders binding to feeder cells, preferably mouse feeder cells, comprise
of binders which bind
to the same epitope than GF 285 (H type 2). In a preferred embodiment, an
antibody binds to
Fuca2Ga104G1cNAc, Fuca2Gal(33(Fuca4)G1cNAc, Fuca2Ga1(34(Fuc(x3)G1cNAc epitope.
A more
preferred antibody comprises of the antibody of clone B389 (DM3014) by Acris.
This epitope is
suitable and can be used to detect, isolate and evaluate of feeder cells,
preferably mouse feeder cells
in culture with human embryonic stem cells. The detection can be performed in
vitro, for FACS
purposes and/or for cell lineage specific purposes. This antibody can be used
to positively isolate
and/or separate and/or enrich feeder cells (negatively select stem cells),
preferably mouse
embryonic feeder cells from a mixture of cells comprising feeder and stem
cells.
Other binders binding to stem cells, preferably human stem cells, comprise of
binders which bind to
the same epitope than GF 289 (Lewis y). In a preferred embodiment, an antibody
binds to
Fuca2Ga1(34(Fuc(x3)GlcNAc epitope. A more preferred antibody comprises of the
antibody of
clone A70-C/C8 (MAB-S201) by Glycotope. This epitope is suitable and can be
used to detect,
isolate and evaluate of stem cells, preferably human stem cells in culture
with feeder cells. The
detection can be performed in vitro, for FACS purposes and/or for cell lineage
specific purposes.
This antibody can be used to positively isolate and/or separate and/or enrich
stem cells (negatively
select feeder cells), preferably human stem cells from a mixture of cells
comprising feeder and stem
cells.
The staining intensity and cell number of stained stem cells, i.e. glycan
structures of the present
invention on stem cells indicates suitability and usefulness of the binder for
isolation and
differentiation marker. For example, low relative number of a glycan structure
expressing cells may
indicate lineage specificity and usefulness for selection of a subset and when
selected/isolated from
the colonies and cultured. Low number of expression is less than 5%, less than
10%, less than 15%,
less than 20%, less than 30% or less than 40%. Further, low number of
expression is contemplated
when the expression levels are between 1-10%, 10%-20%, 15-25%, 20-40%, 25-35%
or 35-50%.
Typically, FACS analysis can be performed to enrich, isolate and/or select
subsets of cells
expressing a glycan structure(s).
High number of glycan expressing cells may indicate usefulness in
pluripotency/multipotency
marker and that the binder is useful in identifying, characterizing, selecting
or isolating pluripotent
or multipotent stem cells in a population of mammalian cells. High number of
expression is more
than 50%, more preferably more than 60%, even more preferably more than 70%,
and most
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preferably more than 80%, 90 or 95%. Further, high number of expression is
contemplated when
the expression levels are between 50-60, 55%-65%, 60-70%, 70-80, 80-90%, 90-
100 or 95-100%.
Typically, FACS analysis can be performed to enrich, isolate and/or select
subsets of cells
expressing a glycan structure(s).
The epitopes recognized by the binders GF 279, GF 287, and GF 289 and the
binders are
particularly useful in characterizing pluripotency and multipotency of stem
cells in a culture. The
epitopes recognized by the binders GF 283, GF 284, GF 286, GF 288, and GF 290
and the binders
are particularly useful for selecting or isolating subsets of stem cells.
These subset or
subpopulations can be further propagated and studied in vitro for their
potency to differentiate and
for differentiated cells or cell committed to a certain differentiation path.
The percentage as used herein means ratio of how many cells express a glycan
structure to all the
cells subjected to an analysis or an experiment. For example, 20% stem cells
expressing a glycan
structure in a stem cell colony means that a binder, eg an antibody staining
can be observed in about
20% of cells when assessed visually.
In colonies a glycan structure bearing cells can be distributed in a
particular regions or they can be
scattered in small patch like colonies. Patch like observed stem cells are
useful for cell lineage
specific studies, isolation and separation. Patch like characteristics were
observed with GF 283, GF
284, GF 286, GF 288, and GF 290.
For positive selection of feeder cells, preferably mouse feeder cells, most
preferably embryonic
fibroblasts, GF 285 is useful. This antibody has lower specificty and may have
binding to e.g.
Lewis y, which has been observed also in mEF cells. It stains almost all
feeder cells whereas very
little if at all staining is found in stem cells. The antibody was however
under optimized condition
revealed to bind to thin surface of embryonic bodies, this was in
complementary to Lewis y
antibody to the core of embryoid body. For all percentages of expression in
immunohistochemical
analysis, see Table 48.
The FACS data in Tables 18, 46-47 and Figure 32 indicates some antibodies
recognizing the major
elongated glycan structure epitopes according to the invention on cell
surfaces. The invention is
especially directed to the use of the H type II, H type I, type I LacNAc
(Lewis c) and globotriose
specific antibodies for the recognition of the embryonic stem cells, GF286,
GF287, GF 279 and
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GF367. The invention is further directed to the major cell populations
isolatable by the antibodies.
The invention is further directed to the antibodies with similar specificties
as the antibodies
recognizing the major cell population of the embryonal stem cells. The
invention is preferably
directed to recognition of the elongated epitopes of H type II and H type I
and type I LacNAc
structures according to the invention by specific binder regents, preferably
by antibodies. The
invention is further directed to the recognition of the novel stem cell marker
globotriose from the
embryonal type stem cells and isolation of the cell popultion by the by using
the specific binder for
the glycan structure.
The invention is in a preferred embodiment directed to the short globoseries
structures such as
globotriose non-reducing end globotriose (Gb3) epitopes: Gala4Ga1, Gala4Ga1(3
and
Gala4Ga1(34G1c for the methods according to the invention. In a preferred
embodiment the
invention is directed to the recognition of the ceramide linked globotriose
epitope. It is realized that
though larger globoseries structures SSEA-3 and SSEA-4 has been indicated from
embryonic stem
cells, this structure has not been known from embryonic type stem cells and
their amounts have
been unpredictable.
Novel methods for recognition of hESC differentiation stage derived from the
factor analyses
Here, statistical analysis was used to identify indicative glycan signals,
glycan structures, and
glycan structure groups for specific recognition of hESC and differentiated
cells. The inventors
revealed that by factor analysis several differentially regulated glycan
groups could be identified
among the N-glycan profiles of hESC and differentiated cells (embryoid bodies
and stage 3
differentiated cells). According to the invention, the cell's differentiation
stage can be assessed by
both positively and negatively selective glycan structures and glycan
structure groups, preferably by
those described above. Specifically, the factor analysis revealed novel
advantageous combinations
of positively+positively, positively+negatively, and negatively+negatively
selective glycan
structures for recognition of the differentiation stage of hESC.
The present invention is specifically directed to performing such analysis by
direct analysis of the
glycan profiles of hESC and differentiated cells, preferably by mass
spectrometry according to the
present invention, the novel added benefit being more effective and reliable
interpretation of the
analysis result.
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In a further embodiment of the present invention, cells in a specific
differentiation stage are
recognized by a glycan structure specific binding reagent, and further
specificity can be gained by
selecting the reagent according to the revealed cell type specificities of the
recognized glycan
groups. The present invention is specifically directed to selected binding
reagents according to the
invention, when the selection is guided by the analysis results described
above. The invention is
further specifically directed to using combinations of binding reagents
selected based on selectivity
of glycan structures revealed in the present invention.
In a further embodiment, the positively and negatively selective binding
reagents are selected based
on the Tables 50 and 51, respectively.
For example, novel beneficial combinations for recognition of hESC
differentiation stage is
selection of at least two specific binding reagents recognizing glycan
structures in at least two
different glycan structure groups of Tables 50 and 51. An even more beneficial
combination for
specific recognition is selection of at least two specific binding reagents
recognizing glycan
structures, at least one in each Table.
The binding reagents selected specifically recognizes at least one preferred
elongated glycan
epitopes according to the invention. More preferably preferred elongated N-
glycan epitopes,
preferably (32Man-epitopes, even more preferably elongated type II LacNAc,
sialylated and
fucosylated derivatives thereof including Lewis x, H type II, and sialyl-Lewis
x. The invention is
further directed to reagents recognizing terminal mannose epitopes of the high
and low mannose
glycans identified.
EXAMPLES
EXAMPLE 1. Analysis of the human embryonic stem cell N-glycome
Structural proposals for N-glycan signals characterized by m/z values as the
other Tables of the
present invention, is presented in Tables 12 and 13. The N-glycan schematic
structures are
according to the recommendations of the Consortium for Functional Glycomics
(www.functionalglycomics.org) and as described e.g. in Goldberg et al. (2005)
Proteomics 5, 865-
875.
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Materials and Methods
Human embryonic stem cell lines (hESC) - Generation of the Finnish hESC lines
FES 21, FES 22, FES 29,
and FES 30 has been described (17) and they were cultured according to the
previous report. Briefly, two of
the analysed cell lines were initially derived and cultured on mouse embryonic
fibroblast (MEF) feeders,
and two on human foreskin fibroblast (HFF) feeder cells. For the present
studies all of the lines were
transferred on HFF feeder cells and cultured in serum-free medium supplemented
with Knockout serum
replacement (Gibco). To induce the formation of embryoid bodies (EB) the hESC
colonies were first allowed
to grow for 10-14 days whereafter the colonies were cut in sma11 pieces and
transferred on non-adherent Petri
dishes to form suspension cultures. The formed EBs were cultured in suspension
for the next 10 days in
standard culture medium without bFGF. For further differentiation (into stage
3 differentiated cells) EB were
transferred onto gelatin-coated culture dishes in media supplemented with
insulin-transferrin-selenium and
cultured for 10 days.
For glycan analysis, the cells were collected mechanically, washed, and stored
frozen until the analysis. In
fluorescence-assisted cell sorting (FACS) analyses 70-90 % of cells from
mechanically isolated hESC
colonies were typically Tra 1-60 and Tra 1-81 positive (not shown). The
differentiation protocol favors the
development of neuroepithelial cells while not directing the differentiation
into distinct terminally
differentiated cell types (18). Stage 3 cultures consisted of a heterogenous
population of cells dominated by
fibroblastoid and neuronal morphologies.
Glycan isolation - Asparagine-linked glycans were detached from cellular
glycoproteins by F.
meningosepticum N-glycosidase F digestion (Calbiochem, USA) essentially as
described (19). Cellular
contaminations were removed by precipitating the glycans with 80-90% (v/v)
aqueous acetone at -20 C and
extracting them with 60% (v/v) ice-cold methanol (20). The glycans were then
passed in water through Cig
silica resin (BondElut, Varian, USA) and adsorbed to porous graphitized carbon
(Carbograph, Alltech, USA)
(21). The carbon column was washed with water, then the neutral glycans were
eluted with 25% acetonitrile
in water (v/v) and the sialylated glycans with 0.05% (v/v) trifluoroacetic
acid in 25% acetonitrile in water
(v/v). Both glycan fractions were additionally passed in water through strong
cation-exchange resin (Bio-
Rad, USA) and Clg silica resin (ZipTip, Millipore, USA). The sialylated
glycans were further purified by
adsorbing them to microcrystalline cellulose in n-butanol:ethanol:water
(10:1:2, v/v), washing with the same
solvent, and eluting by 50% ethanol:water (v/v). All the above steps were
performed on miniaturized
chromatography columns and small elution and handling volumes were used.
Mass spectrometry and data analysis - MALDI-TOF mass spectrometry was
performed with a Bruker
Ultraflex TOF/TOF instrument (Bruker, Germany) essentially as described (22).
Relative molar abundancies
of neutral and sialylated glycan components can be accurately assigned based
on their relative signal
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intensities in the mass spectra when analyzed separately as the neutral and
sialylated N-glycan fractions (22-
25). Each step of the mass spectrometric analysis methods was controlled for
reproducibility by mixtures of
synthetic glycans or glycan mixtures extracted from human cells.
The mass spectrometric raw data was transformed into the present glycan
profiles by carefully removing the
effect of isotopic pattern overlapping, multiple alkali metal adduct signals,
products of elimination of water
from the reducing oligosaccharides, and other interfering mass spectrometric
signals not arising from the
original glycans in the sample. The resulting glycan signals in the presented
glycan profiles were normalized
to 100% to allow comparison between samples.
Quantitative difference between two glycan profiles (%) was calculated
according to Equation 1:
n
difference = ~ I pi,. - pi,b , (1)
_,
wherein p is the relative abundance (%) of glycan signat i in profile a or b,
and n is the total number of
glycan signals.
Relative difference between a glycan feature in two profiles was calculated
according to Equation 2:
x
relative difference = x P , (2)
b
wherein P is the sum the relative abundancies of the glycan signals with the
glycan feature in profile a or b, x
is 1 when a> b, and x is -1 when a < b.
The glycan analysis method was validated by subjecting human cell samples to
blinded analysis by five
different persons. The results were highly comparable (data not shown),
especially by the terms of detection
of individual glycan signals and their relative signal intensities, showing
that the present method reliably
produced glycan profiles suitable for comparision of analysis results from
different cell types.
Glycosidase analysis - The neutral N-glycan fraction was subjected to
digestion with Jack bean a-
mannosidase (Canavalia ensiformis; Sigma, USA) essentially as described (22).
NMR methods - For NMR spectroscopic analyses, larger amounts of hESC were
grown on mouse feeder cell
(MEF) layers. The isolated glycans were purified for the analysis by gel
filtration high-pressure liquid
chromatography in a column of Superdex peptide HR 10/30 (Amersham), with water
(neutral glycans) or 50
mM NH4HCO3 (sialylated glycans) as the eluant at a flow rate of 1 ml/min. The
eluant was monitored at 214
nm, and oligosaccharides were quantified against external standards. The
amount of N-glycans in NMR
analysis was below five nanomoles. Prior to NMR analysis the purified glycome
fractions were repeatedly
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dissolved in 99.996% deuterium oxide and dried to omit H20 and to exchange
sample protons. The proton
NMR spectra at 800 MHz were recorded using a cryo-probe for enhanced
sensitivity.
Statistical procedures - Glycan score distributions of all three
differentiation stages (hESC, EB, and stage 3
differentiated cells) were analyzed by the Kruskal-Wallis test. Pairwise
comparisons were performed by the
2-tailed Student's t-test with Welch's approximation and 2-tailed Mann-Whitney
U test. A p value less than
0.05 was considered significant. The statistical analyses are described in
more detail in Supplementary data.
Lectin staining - Fluorescein-labelled lectins used in lectin histochemistry
were from EY Laboratories
(USA). Specificity of binding was controlled by inhibition experiments with
a3'-sialyllactose and D-
mannose for Maackia amurensis agglutinin (MAA) and Pisum sativum agglutinin
(PSA), respectively.
Results
In order to generate mass spectrometric glycan profiles of hESC, embryoid
bodies (EB), and further
differentiated cells, a matrix-assisted laser desorption-ionization (MALDI-
TOF) mass spectrometry based
analysis was performed. We focused on the most common type of protein post-
translational modifications,
N-glycans, which were enzymatically released from cellular glycoproteins.
During glycan isolation and
purification, the total N-glycan pool was separated by an ion-exchange step
into neutral N-glycans and
sialylated N-glycans. These two glycan fractions were then analyzed separately
by mass spectrometric
profiling (Fig. 2), which yielded a global view of the N-glycan repertoire.
Over one hundred N-glycan
signals were detected from each cell type demonstrating that N-glycosylation
is equally sophisticated in stem
cells and cells differentiated from them. The proposed monosaccharide
compositions corresponding to the
detected masses of each individual signal in Figure 2 are indicated by letter
code. However, it is important to
realize that many of the mass spectrometric signals in the present analyses
include multiple isomeric
structures and the one hundred most abundant signals very likely represent
hundreds of different molecules.
The relative abundances of the observed glycan signals were determined based
on their relative signal
intensities (22,24-25), which allowed analysis of N-glycan profile differences
between samples. The present
data demonstrate that mass spectrometric profiling can be used in effective
quantitative comparison of total
glycan profiles, especially to pin-point the major glycosylation differences
between related samples. In the
following, we have expressed relative abundancies of glycan signals as molar
proportions of the total
detected N-glycans. However, these figures should be recognized as practical
approximations based on the
present data instead of absolutely quantitative percentages of the N-glycome.
In most of the previous glycomic studies of mammalian cells and tissues the
isolated glycans have been
derivatized (permethylated) prior to mass spectrometric profiling (26-29) or
chromatographic analysis (30).
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However, we chose to directly analyze the picomolar quantities of unmodified
glycans and increased
sensitivity was achieved by omitting the derivatization and the subsequent
additional purification steps. Our
glycan purification scheme enabled N-glycan profiling analysis from samples as
small as 100 000 cells
showing that sensitivity of the analysis step is not a limiting factor in
glycomic studies with scarce biological
samples.
Overview of the hESC N-glycome: Neutral N-glycans Neutral N-glycans comprised
approximately two thirds
of the combined neutral and sialylated N-glycan pools of hESC. The 50 most
abundant neutral N-glycan
signals detected in the four hESC lines are presented in Figure 2A (blue
columns). The similarity of the
profiles, which is indicated by the minor variation in the glycan signals,
suggests that the four cell lines
closely resemble each other. For example, 15 of the 20 most abundant glycan
signals were the same in every
hESC line. These 15 neutral N-glycan signals characteristic of the hESC N-
glycome are listed in Table 7.
The five most abundant signals (H5N2, H6N2, H-,Nz, H8N2, and H9N2; for
abbreviations see Fig. 2) comprised
76% of the neutral N-glycans of hESC and dominated the profile.
Sialylated N-glycans - All N-glycan signals in the sialylated N-glycan
fraction (Fig. 2B, blue columns)
contained sialic acid residues (S: N-acetylneuraminic acid, or G: N-
glycolylneuraminic acid). There was
more variation between individual cell lines in the 50 most abundant
sialylated N-glycans than in the neutral
N-glycans. However, the four eell lines again resembled each other. The five
most abundant sialylated N-
glycan signals were the same in every cell line: S1H5N4Fi, S1H5N4F2, S2H5N4Fi,
SiHsN4, and SiH6N5F1. The
15 sialylated N-glycan signals common to all the hESC lines are listed in
Table 7.
The most abundant sialylated glycan signals contained the H5N4 core
composition and differed only by
variable number of sialic acid (S or G) and deoxyhexose (F) residues. These
comprised 61 % of the total
glycan signal intensity in Figure 2B. Similarly, another common core structure
was H6N5 that was present in
seven signals comprising 12% of the total glycan signal intensity. These
examples highlight the biosynthetic
mechanism that leads to the complex spectra of N-glycan structures in cells: N-
glycans typically consist of
common core structures that are modified by the addition of variable epitopes
(Fig. 3A).
Importantly, we detected N-glycans containing N-glycolylneuraminic acid (G) in
the hESC samples, for
example glycans G1H5N4, G1S1H5N4, and G2H5N4. N-glycolylneuraminic acid has
previously been reported
in hESC as an antigen transferred from culture media containing animal-derived
materials (31). Accordingly,
the serum replacement medium used in the present experiments contained bovine
serum proteins. We have
recently detected Neu5Gc in N-glycans of hESC and in vitro cultured human
mesenchymal stem cells by
mass spectrometric N-glycan analysis (32).
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Variation between individual cell lines - Although the four hESC lines shared
the same overall N-glycan
profile, there was cell line specific variation within the profiles.
Individual glycan signals unique to each cell
line were detected, indicating that every cell line was slightly different
from each other with respect to the
approximately one hundred most abundant N-glycan structures. Importantly, the
30 most common N-glycan
signals in all the hESC lines accounted for circa 85% of the total detected N-
glycans, and they represent a
useful approximation of the hESC N-glycome (Table 7).
Transformation of the N-glycome during hESC differentiation - A major goal of
the present study was to
identify glycan structures that would be specific to either stem cells or
differentiated cells, and could
therefore serve as differentiation stage markers. In order to determine
whether the hESC N-glycome
undergoes changes during differentiation, the N-glycan profiles obtained from
hESC, EB, and stage 3
differentiated cells were compared (Fig. 2). The profiles of the
differentiated cell types (EB and stage 3
differentiated cells) were clearly different compared to the profiles of
undifferentiated hESC, as indicated by
non-overlapping distribution bars in many glycan signals. Further, there were
many signals present in both
hESC and EB that were not detected in stage 3 differentiated cells. Overall,
10% of the glycan signals
present in hESC had disappeared in stage 3 differentiated cells.
Simultaneously numerous new signals
appeared in EB and stage 3 differentiated cells. The proportion of these
differentiation-associated N-glycan
signals in EB and stage 3 differentiated cells was 14% and 16%, respectively.
Taken together, differentiation induced the appearance of new N-glycan types
while earlier glycan types
disappeared. Further, we found that the major hESC-specific N-glycosylation
features were not expressed as
discrete glycan signals, but instead as glycan signal groups that were
characterized by specific
monosaccharide composition features. In other words, differentiation of hESC
into EB induced the
disappearance of not only one but multiple glycan signals with hESC-associated
features, and simultaneously
also the appearance of glycan signal groups with other, differentiation-
associated features.
The N-glycan profiles of the differentiated cells were also quantitatively
different from the undifferentiated
hESC profiles. A practical way of quantifying the differences between glycan
profiles is to calculate the sum
of the signal intensity differences between two samples (see Experimental
procedures, Equation 1).
According to this method, the EB neutral and sialylated N-glycan profiles had
undergone a quantitative
change of 14% and 29% from the hESC profiles, respectively. Similarly, the
stage 3 differentiated cell
neutral and sialylated N-glycan profiles had changed by 15% and 43%,
respectively. Taking into account that
the proportion of sialylated to neutral N-glycans in hESC was approximately
1:2, the total N-glycan profile
change was approximately 25% during the transition from hESC to stage 3
differentiated cells.
The present data indicated that the mass spectrometric profile of the hESC N-
glycome consisted of two
discrete parts regarding propensity to change during hESC differentiation - a
constant part of circa 75% and
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a changing part of circa 25%. In order to characterize the associated N-glycan
structures, and to identify the
potential biological roles of the constant and changing parts of the N-
glycome, we performed structural
analyses of the isolated hESC N-glycan samples.
Structural analyses of the major hESC N-glycans: Preliminary structure
assignment based on
monosaccharide compositions - Human N-glycans can be divided into biosynthetic
groups of high-mannose
type, hybrid-type, and complex-type N-glycans (33-34). Due to abundant
expression of mannosylated N-
glycans smaller than the classical high-mannose type structures in hESC, we
added a new group called low-
mannose N-glycans into this classification. To determine the presence of these
N-glycan groups in the cells,
assignment of probable structures matching the monosaccharide compositions of
each individual signal was
performed utilizing the established pathways of human N-glycan biosynthesis.
Here, the detected N-glycan
signals were classified into four N-glycan groups according to the number of N
and H residues in the
proposed compositions as shown in Figure 3A: 1) high-mannose type and 2) low-
mannose type N-glycans,
which are both characterized by two N residues (N-2), 3) hybrid-type or
monoantennary N-glycans, which
are classified by three N residues (N-3), and 4) complex-type N-glycans, which
are characterized by four or
more N residues (N>4) in their proposed monosaccharide compositions. However,
this is an approximation
and in addition to complex-type N-glycans also hybrid-type or monoantennary N-
glycans may contain more
than three N residues.
The data was analyzed quantitatively by calculating the percentage of glycan
signals in the total N-glycome
belonging to each structure group (Table 3) and comparing the hESC and
differentiated cell glycan
classification data (Fig. 3B). The relative differences in the structural
groups reflect the activities of different
biosynthetic pathways in each cell type. For example, the proportion of hybrid-
type or monoantennary N-
glycans was increased when hESC differentiated into EB, indicating that
different glycan biosynthesis routes
were favored in EB than in hESC. However, no glycan structure classes
disappeared or appeared in the hESC
differentiation process, which indicated that the fundamental N-glycan
biosynthesis routes were not changed
during differentiation. The proportion of low-mannose type N-glycans was
surprisingly high in the light of
earlier published studies of human N-glycosylation. However, according to our
studies this is not specific to
hESC (T. Satomaa, A. Heiskanen, J. Natunen, J. Saarinen, N. Salovuori, A.
Olonen, J. Helin, M. Blomqvist,
0. Carpen, unpublished results).
Verification of structure assignments by enzymatic glycan degradation and
nuclear magnetic resonance
spectroscopy - In order to validate the glycan structure assignments made
based on the mass spectrometric
analysis and the proposed monosaccharide compositions, we performed enzymatic
degradation and proton
NMR spectroscopy analyses of selected neutral and sialylated N-glycans.
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For the validation of neutral N-glycans we chose the glycans H5N2, H6N2, H7N2,
HgNz, and HgNz, which were
the most abundant N-glycans in all studied cell types (Fig. 2A). The
monosaccharide compositions of these
glycans had already suggested (Fig. 3A) that they were high-mannose type N-
glycans (33). To test this
hypothesis, neutral N-glycans from hESC and the differentiated cell samples
were treated with a-
mannosidase, and analyzed both before and after the enzymatic treatment by
MALDI-TOF mass
spectrometry (data not shown). The glycans in question were degraded and the
corresponding signals
disappeared from the mass spectra, indicating that they had contained a-linked
mannose residues.
The neutral N-glycan fraction was further analyzed by nanoscale proton NMR
spectroscopy. In the obtained
NMR spectrum of the hESC neutral N-glycans signals consistent with high-
mannose type N-glycans were
abundant (Fig. 4A and Table 8), supporting the conclusion that they were the
major glycan components in
the sample. In proton NMR spectroscopic analysis of the sialylated N-glycan
fraction, N-glycan backbone
signals consistent with biantennary complex-type N-glycans were the major
detected signals (Fig. 4B and
Table 9), in line with the preliminary assignment made based on the proposed
monosaccharide compositions.
The present results indicated that the classification of the glycan signals
within the total N-glycome data
could be used to construct an approximation of the whole N-glycome.
Complex fucosylation of N-glycans is characteristic of hESC - Differentiation
stage associated changes in
the sialylated N-glyean profile of hESC were more drastic than in the neutral
N-glycan fraction and the
group of five most abundant sialylated N-glycan signals was different at every
differentiation stage (Fig. 2B).
In particular, there was a significant differentiation-associated decrease in
the relative amounts of glycans
S1HsN4Fz and S1H5N4F3 as well as other glycan signals that contained at least
two deoxyhexose residues
(F>2). In contrast, glycan signals such as S2H5N4 that contained no F were
increased in the differentiated cell
types. The results suggested that sialylated N-glycans in undifferentiated
hESC were subject to more
complex fucosylation than in the differentiated cell types (Fig. 3B). The most
common fucosylation type in
human N-glycans is a1,6-fucosylation of the N-glycan core structure (35). The
NMR analysis of the
sialylated N-glycan fraction of hESC also revealed al,6-fucosylation of the N-
glycan core as the most
abundant type of fucosylation (Table 9). In N-glycans containing more than one
fucose residue there has to
be other fucose linkages in addition to the al,6-linkage (35). The F>2
stractural feature decreased as the cells
differentiated, indicating that complex fucosylation was characteristic of
undifferentiated hESC.
N-glycans with terminal N-acetylhexosamine residues become more common with
differentiation - A major
group of N-glycan signals which increased during differentiation contained
equal amounts of N-
acetylhexosamine and hexose residues (N H) in their monosaccharide composition
(e.g. S1H5N5Fi). This
was consistent with N-glycan structures containing non-reducing terminal N-
acetylhexosamine residues
since such complex-type N-glycans generally have monosaccharide compositions
of either N-H or N>H
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(Fig. 3A). EB and stage 3 differentiated cells showed increased amounts of
potential terminal N-
acetylhexosamine structures (Fig. 3B).
Glycome profiling can identify the differentiation stage of hESC - The glycome
profile analyses indicated
that the studied hESC lines and differentiated cells had differentiation stage
specific N-glycosylation
features. However, the data also demonstrated variation between individual
cell lines. To test whether the
obtained N-glycan profiles could be used to generate an efficient
discrimination algorithm that would
discriminate between hESC and differentiated cells, we performed a statistical
evaluation of the mass
spectrometric data (see Supplementary data for details). The results are
described graphically in Figure 5.
The differentiated cell samples (EB and stage 3 differentiated cells) were
significantly discriminated from
hESC with p < 0.01. The stage 3 differentiated cell samples were also
significantly separated from the EB
samples with p < 0.01. This suggested that the hESC N-glycan profiles were
similar at the glycome level
despite of individual differences at the level of individual glycan signals.
The result also suggested that
glycome profiling is a potential tool for monitoring the differentiation
status of stem cells.
The identified hESC glycans can be targeted at the cell surface - From a
practical perspective stem cell
research would be best served by reagents that recognize cell-type specific
target structures on cell surface.
To investigate whether individual glycan structures we had identified would be
accessible to reagents
targeting them at the cell surface we performed lectin labelling of two
candidate structure types. Lectins are
proteins that recognize glycans with specificity to certain glycan structures
also in hESC (36-37). hESC
colonies grown on mouse feeder cell layers were labeled in vitro by
fluorescein-labelled lectins (Fig. 6). The
hESC cell surfaces were clearly labeled by Maackia amurensis agglutinin (MAA)
that recognizes structures
containing a2,3-linked sialic acids, indicating that sialylated glycans were
abundant on the hESC cell surface
(Fig. 6A). Such glycans would thus be available for recognition by more
specific glycan-recognizing
reagents such as antibodies. In contrast, the cell surfaces were not labelled
by Pisum sativum agglutinin
(PSA) that recognizes a-mannosylated glycans (Fig. 6B). However, PSA labelled
the cells after
permeabilization (data not shown), suggesting that the majority of the
mannosylated N-glycans in hESC
were localized in intracellular cell compartments such as ER or Golgi (Fig.
6C). Interestingly, the mouse
fibroblast cells showed complementary staining patterns compared to hESC,
suggesting that these lectin
reagents efficiently discriminated between hESC and feeder cells. Together the
results suggested that the
glycan structures we identified could be utilized to design reagents
specifically targeting undifferentiated
hESC.
Discussion
In the present study, novel glycan analysis methods were applied in the first
structural analysis of hESC N-
glycan profiles. By employing efficient purification of non-derivatized
glycans we demonstrated mass
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spectrometric N-glycan profiles of the scarce hESC and differentiated cell
samples from approximately
100 000 cells. As a result, dramatic glycan profile differences were
discovered between the analyzed cell
types. The objective in the present study was to provide a global view on the
N-glycome profile, or a
"fingerprint" of hESC N-glycosylation, rather than to present the stem cell
glycome in terms of the molecular
structures of each glycan component. The structural information already
allowed us to determine the most
abundant N-glycan structures of hESC. Furthermore, changes observed in the N-
glycan profiles provided
vast amount of information regarding hESC N-glycosylation and its changes
during differentiation, allowing
rational design of detailed structural studies of selected glycan components.
It will be of great interest to
apply these glycan analysis methods to other stem cell and differentiated cell
types.
The results indicated that a defined group of N-glycan signals dominates the
hESC N-glycome forming a
unique stem cell glycan profile. For example, the fifteen most abundant
neutral N-glycan signals and fifteen
most abundant sialylated N-glycan signals in hESC together comprised over 85%
of the N-glycome. On the
other hand, structurally different glycan structures were favored during hESC
differentiation. This suggests
that N-glycan biosynthesis in hESC is a controlled and predetermined process.
Based on our results the hESC N-glycome seems to contain both a constant part
consisting of "housekeeping
glycans", and a changeable part that is altered when the hESC differentiate
(Fig. 2). The constant part seems
to contain mostly high-mannose type and biantennary complex-type N-glycans,
which may need to be
present at all times for the maintenance of fundamental cellular processes.
Significantly, 25% of the total N-
glycan profile of hESC changed during their differentiation (see Supplementary
Fig. S4). This indicates that
during differentiation hESC dramatically change both their appearance towards
their environment and
possibly also their own capability to sense and respond to exogenous signals.
Our data show that the differentiation-associated change in the N-glycome was
mostly generated by the
addition or removal of variable epitopes on similar N-glycan core
compositions. The present lectin staining
experiments demonstrated that sialylated glycans were abundant on the cell
surface of hESC, indicating that
cell type specific N-glycan structures are potential targets for development
of more specific recognition
reagents. It seems plausible that knowledge of the changing surface glycan
epitopes could be utilized as a
basis in developing reagents and culture systems that would allow improved
identification, selection,
manipulation, and culture of hESC and their progeny.
Protein-linked glycans perform their functions in cells by acting as ligands
for specific glycan receptors (38-
39), functioning as structural elements of the cell (40), and modulating the
activity of their carrier proteins
and lipids (2). More than half of all proteins in a human cell are
glycosylated. Consequently, a global change
in protein-linked glycan biosynthesis can simultaneously modulate the
properties of multiple proteins. It is
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likely that the large changes in N-glycans during hESC differentiation have
major influences on a number of
cellular signaling cascades and affect in profound fashion biological
processes within the cells.
The major hESC specific glycosylation feature we identified was the presence
of more than one deoxyhexose
residue in N-glycans, indicating complex fucosylation. Fucosylation is known
to be important in cell
adhesion and signalling events as well as being essential for embryonic
development (41). Knock-out of the
N-glycan core a1,6-fucosyltransferase gene FUT8 leads to postnatal lethality
in mice (42), and mice
completely deficient in fucosylated glycan biosynthesis do not survive past
early embryonic development
(43).
Fucosylated glycans such as the SSEA-1 antigen (7, 44-45) have previously been
associated with both mouse
embryonic stem cells (mESC) and human embryonic carcinoma cells (EC; 16), but
not with hESC. The
published gene expression profiles for the same hESC lines as studied here
(46) have demonstrated that three
human fucosyltransferase genes, FUTI, FUT4, and FUT8 are expressed in hESC,
and that FUTI and FUT4
are overexpressed in hESC when compared to EB. FUT8 encodes the N-glycan core
a1,6-fucosyltransferase
whose product was identified as the major fucosylated epitope in hESC N-
glycans (Fig. 4B). The hESC-
specific expression ofFUTI and FUT4, encoding for a1,2-fucosyltransferase and
a1,3-fucosyltransferase
enzymes (47), respectively, correlate with our findings of simple fucosylation
in EB and complex
fucosylation in hESC. Interestingly, the FUT4-encoded enzyme is capable of
synthesizing the SSEA-1
antigen (48-49). Although hESC do not express the specific glycolipid antigen
recognized by the SSEA-1
antibody, they share with mESC the characteristic feature of complex
fucosylation and may also share the
conserved essential biological functions of fucosylated glycan epitopes.
New N-glycan forms also emerged in EB and stage 3 differentiated cells. These
structural features included
additional N-acetylhexosamine residues, potentially leading to new N-glycan
terminal epitopes. Another
differentiation-associated feature was increase in the molar proportions of
hybrid-type or monoantennary N-
glycans. Biosynthesis of hybrid-type and complex-type N-glycans has been
demonstrated to be biologically
significant for embryonic and postnatal development in the mouse (50-51). The
preferential expression of
complex-type N-glycans in hESC and then the change in the differentiating EB
to express more hybrid-type
or monoantennary N-glycans may be significant for the process of stem cell
differentiation.
Human embryonic stem cell lines have previously been demonstrated to have a
common genetic stem cell
signature that can be identified using gene expression profiling techniques
(17,52-54). Such signatures have
been proposed to be useful in hESC characterization. In the present report we
provide the first glycomic
signatures for hESC. The profile of the expressed N-glycans might be a useful
tool for analyzing and
elassifying the differentiation stage in association with gene and protein
expression analyses. Here we
demonstrated that a glycan score algorithm was able to reliably differentiate
the cell samples in separate
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differentiation stages (Fig. 5). Glycome profiling might be more sensitive
than the use of any single cell
surface marker and especially useful for the quality control of hESC-based
cell products. However, further
analysis of the hESC glycome may also lead to discovery of novel glycan
antigens that could be used as stem
cell markers in addition to the commonly used SSEA and Tra glycan antigens.
In conclusion, hESC have a unique N-glycome which undergoes major changes when
the cells differentiate.
Information regarding the specific glycan structures may be utilized in
developing reagents for targeting
these cells and their progeny. Future studies investigating the developmental
and molecular regulatory
processes resulting in the observed N-glycan profiles may provide significant
insight into mechanisms of
human development and regulation of glycosylation.
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Yamaguchi, Y., Aze, Y., Tomiyama,
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D.J., Homeister, J.W., and
Lowe, J.B. (2002) J. Cell Biol. 158, 801-815
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44. Solter, D., and Knowles, B.B. (1978) Proc. Natl. Acad. Sci. U.S.A. 75,
5565-5569
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M.S., and Puri, R.K. (2004) Blood
103,2956-2964
EXAMPLE 2. Analysis of N-glycan composition groups with terminal HexNAc in
stem cells
and differentiated cells.
Methods. To analyze the presence of terminal HexNAc containing N-glycans
characterized by the
formulae: nHeXNAc = nxeX >_ 5 and naxeX > 1(group I), and to compare their
occurrence to terminal
HexNAc containing N-glycans characterized by the formulae: nxeXNAc = nxeX > 5
and ndHeX = 0
(group II), N-glycans were isolated, purified and analyzed by MALDI-TOF mass
spectrometry as
described in the preceding Examples. They were assigned monosaccharide
compositions and their
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relative proportions within the obtained glycan profiles were determined by
quantitative profile
analysis as described above. The following glycan signals were used as
indicators of the specific
glycan groups (monoisotopic masses):
Ia, HexsHexNAcsdHexi: m/z for [M+Na]+ ion 2012.7
Ib, NeuAciHexSHexNAcsdHexi: m/z for [M-H]- ion 2279.8
Ic, NeuAc2Hex5HexNAc5dHexl: m/z for [M-H]- ion 2570.9
Id, NeuAclHexSHexNAcsdHexz: m/z for [M-H]- ion 2425.9
IIa, NeuAciHexSHexNAcs: m/z for [M-H]- ion 2133.8
Further, relative expression of glycan signals Hex3HexNAc5: m/z for [M+Na]+
ion 1542.6 and
Hex3HexNAc5dHexi: m/z for [M+Na]+ ion 1688.6 was also analyzed.
Results. As an indicator of group I glycans, Ib was detected in various N-
glycan samples isolated
from stem cell samples, including EB and st.3 differentiated cells,.
hESC lines FES 22, FES 29, and FES 30: Ia, Ib, Ic, Id, and IIa were
overexpressed in EB and st.3
when compared to hESC. Specifically, Ia was not expressed in hESC and Ila was
expressed in only
1/3 of the hESC samples. The relative abundance of Hex3HexNAc5 and
Hex3HexNAc5dHexi was
also increased in EB and st.3: for Hex3HexNAc5 by 6.1 fold and 7.8 fold, and
for
Hex3HexNAc5dHexi by 1.2 fold and 2.6 fold for the transitions from hESC to EB
and hESC to st.3,
respectively.
EXAMPLE 3. Evaluation of individual variation in relative proportions of N-
glycan signals of
hESC lines.
The propensity of each glycan signal to be subject to individual variation
between cell lines was
estimated by calculating the average deviation of the glycan signal relative
proportions between the
four hESC lines. The deviations were then evaluated as proportion of average
deviation from the
average signal proportion (in %). In this calculation, three groups of glycan
signals were obtained:
over 100% average deviation (large individual variation), between 50-100%
average deviation
(substantial individual variation), and between 0-50% average deviation
(little individual variation).
Below are the glycan signals listed in Tables 1 and 2 as grouped according to
this.
Over 100% (large individual variation):
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Neutral N-glycans H4N3F2, H5N5, H4N5, H4N5F2, H4N4F2, H6N4, H4N5F1, H5N5F1,
H3N5,
H2N4F1, H4N4, H4N5F3, H2N2, H3N5F1, H5N2F1, and H6N3F1.
Sialylated N-glycans S2H7N6F1, S2H4N3F1, S2H5N5F1, S1H5N5, S3H6N5, S2H6N5F2,
S2H5N3F1, S2H3N3F1, S1H8N7F1, S1H6N4F2, S1H5N3F1, S2H6N4, S1H4N4F1, G2H5N4,
and
S1H6N4F1Ac.
Over 50% (moderate individual variation):
Neutral N-glycans H1N2, H11N2, H5N3F1, H5N4F3, H5N4F2, H3N2F1, N2N2F1, H6N3,
and
H3N2.
SialylatedN-glycans S2H5N4, S1H6N5F3, S2H4N5F1, S1H6N4F1, S1G1H5N4, S1H6N3,
S1H5N3, S1H4N3, S1H7N6F2, G1H5N4, S2H2N3F1, S1H6N5, and S1H7N6F3.
Over 0% (little individual variation):
Neutral N-glycans H5N3, H5N4F1, H6N5F1, H3N3, H3N4F1, H4N2F1, H6N5, H3N3F1,
H4N3,
H4N2, H4N4F1, H5N4, H8N2, H4N3F1, HlON2, H5N2, H7N2, H6N2, and H9N2.
SialylatedN-glycans S1H4N5F2, S1H7N6F1, S1H5N4F3, S1H5N5F2, S1H6N5F2,
S1H4N5F1,
S2H6N5F1, G1H5N4F1, S1H5N4F2, S2H5N4F1, S1H5N5F1, S1H6N5F1, S1H5N4, S1H4N3F1,
and S1H5N4F1.
The major glycan signals were in the group of little individual variation.
This group also included
the major biantennary-size complex-type N-glycans including S1H5N4F1, the
major high-mannose
type N-glycans including H9N2, and the major complex-fucosylated complex-type
N-glycans
including S1H5N4F2 and S1H5N4F3, showing that these major hESC-associated
glycan features
were not subject to significant individual variation between hESC lines.
Cell line specific N-glycan profile data is presented in Tables 10 and 11,
formatted as in Example 1.
EXAMPLE 4. Analysis of N-glycan, Glycolipid and O-glycan cellular glycan types
by specific
glycosidases and mass spectrometry.
Assignment of Lewis x on N-glycans
Previously it was indicated by combination of NMR spectroscopy and (31,4-
galactosidase, (3-N-
acetylglucosaminidase, and (3-hexosaminidase digestions that hESC neutral
monoantennary and
biantennary-size N-glycans preferentially contained type 2 LacNAc antennae and
also minor
amounts of LacdiNAc antennae, more preferentially in a complex-type
biantennary N-glycan
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backbone with (31,2-branches. Here it was studied by al,3/4-fucosidase
digestion of the hESC
neutral N-glycan fraction which specific antennae contained 0,3-fucosylation
decorations of these
antennae. The glycan sample was produced as described in the other Examples of
the present
invention from similar hESC samples.
Monoantennary N-glycans that were digested with a1,3/4-fucosidase included
H4N3F2 (m/z 1590),
digested into H4N3F1 (1444), preferentially including the non-reducing
terminal structure
Lex(32Man, more preferentially also including a complete N-glycan structure
Lex(32Mana3(Mana6)Manp4GlcNAc(34(Fuca6)G1cNAc.
Biantennary-size N-glycans that were digested with al,3/4-fucosidase included
H5N4F2 (m/z 1955)
and H5N4F3 (2101), which were digested into H5N4F1 (1809); and H4N5F2 (1996)
and H4N5F3
(2142), which were digested into H4N5F1 (1850). These glycans preferentially
included the non-
reducing terminal structures Lex(32Man and GaLNAc(34(Fuca3)G1cNAc(32Man,
respectively, more
preferentially also including complete N-glycan structures:
Lex(32Mana3(Lex(32Mana6)Man(34G1cNAc(34(Fuca6)G1cNAc and
GaLNAc(34(Fuca3)G1cNAc(32ManaX(Lex(32ManaY)Man(34G1eNAc(34(Fuca6)GIeNAc,
wherein X
and Y are either 3 or 6, and X# Y.
0-glycan and glycolipid analysis
The glycosphingolipid glycan and reducing O-glycan samples were isolated from
studied cell types,
analyzed by mass spectrometry, and further analyzed by expoglycosidase
digestions combined with
mass spectrometry as described in the present invention and the preceding
Examples. Non-reducing
terminal epitopes were analyzed by digestion of the glycan samples with S.
pneumoniae (31,4-
galactosidase (Calbiochem), bovine testes (3-galactosidase (Sigma), A.
ureafaciens sialidase
(Calbiochem), S. pneumoniae a2,3-sialidase (Calbiochem), S. pneumoniae (3-N-
acetylglucosaminidase (Calbiochem), X. manihotis a1,3/4-fucosidase
(Calbiochem), and a1,2-
fucosidase (Calbiochem). The results were analyzed by quantitative mass
spectrometric profiling
data analysis as described in the present invention. The results with
glycosphingolipid glycans are
summarized in Table 22 including also core structure classification determined
based on proposed
monosaccharide compositions as described in the footnotes of the Table.
Analysis of neutral 0-
glycan fractions revealed quantitative differences in terminal epitope
glycosylation as follows: non-
reducing terminal type 1 LacNAc ((31,3-linked Gal) had above 5% proportion is
characteristic to
hESC.. Fucosylation degree of type 2 LacNAc containing 0-glycan signals at mlz
771
(HexzHexNAcz) and 917 (HexzHexNAczdHexi) was 28% in hESC.
In conclusion, these results from 0-glycans and glycosphingolipid glycans
demonstrated significant
cell type specific differences and also were significantly different from N-
glycan terminal epitopes
within each cell type analyzed in the present invention.
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EXAMPLE 5. Glycosphingolipid glycans of human stem cells.
EXPERIMENTAL PROCEDURES
Samples from hESC grown on mouse fibroblast feeder cells were produced as
described in the
preceding Examples. Neutral and acidic glycosphingolipid fractions were
isolated from cells
essentially as described (Miller-Podraza et al., 2000). Glycans were detached
by Macrobdella
decora endoglycoceramidase digestion (Calbiochem, USA) essentially according
to manuacturer's
instructions, yielding the total glycan oligosaccharide fractions from the
samples. The
oligosaccharides were purified and analyzed by MALDI-TOF mass spectrometry as
described in the
preceding Examples for the protein-linked oligosaccharide fractions.
RESULTS AND DISCUSSION
Human embryonic stem cells (hESC)
hESC neutral lipid glycans. The analyzed mass spectrometric profile of the
hESC glycosphingolipid
neutral glycan fraction was analyzed (not shown).
Structural analysis of the major neutral lipid glycans. The six major glycan
signals, together
comprising more than 90% of the total glycan signal intensity, corresponded to
monosaccharide
compositions Hex3HexNAci (730), Hex3HexNAc1dHex1 (876), HexzHexNAci (568),
Hex3HexNAo2 (933), Hex4HexNAci (892), and HexqHexNAoz (1095).
In P1,4-galactosidase digestion, the relative signal intensities of 1095 and
730 were reduced by
about 30% and 10%, respectively. This suggests that 730 and 1095 contain minor
components with
non-reducing terminal 01,4-Gal epitopes, preferably including the structures
Gal(34G1cNAcLac and
Ga1(34G1cNAc[HexiHexNAci]Lac. The other major components were thus shown to
contain other
terminal epitopes. Further, the glycan signal Hex5HexNAc3 (1460) was digested
to Hex3HexNAc3
(1136), indicating that the original signal contained glycan structures
containing two (31,4-Gal.
The major glycan signals were not sensitive to a-galactosidase digestion.
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In a1,3/4 fucosidase digestion, the signal intensity of 876 was reduced by
about 10%, indicating
that only a minor proportion of the glycan signal corresponded to glycans with
a1,3- or a 1,4-linked
fucose residue. The major affected signal in the total profile was
Hex3HexNAcldHex2 (1022),
indicating that it included glycans with either al,3-Fuc or al,4-Fuc. 511 was
reduced by about
30%, indicating that the signal contained a minor component with al,2-Fuc,
preferentially including
Fuca2Ga1(34Glc (Fuca2'Lac, 2'-fucosyllactose).
When the a1,3/4 fucosidase reaction product was further digested with a1,2
fucosidase, 876 was
completely digested into 730, indicating that the structure of the majority of
the signal intensity
contained non-reducing terminal a1,2-Fuc, preferably including the structure
Fuca2[HexiHexNAci]Lac, more preferably including Fuca2GalHexNAcLac. Another
partly
digested glycan signal was Hex4HexNAc2dHex, (1241) that was thus indicated to
contain al,2-Fuc,
preferably including the structure Fuca2[Hex2HexNAc2]Lac, more preferably
including
Fuca2Ga1[HexlHexNAc2]Lac. 511 was completely digested, indicating that the
original signal
contained a major component with al,3/4-Fuc, preferentially including
Gal(34(Fuca3)Glc (3-
fucosyllactose).
When the a1,3/4 fucosidase and a1,2 fucosidase reaction product was further
digested with /j1,4-
galactosidase, the majority of the newly formed 730 was not digested, i.e. the
relative proportion of
568 was not increased compared to (31,4-galactosidase digestion without
preceding fucosidase
treatments. This indicated that the majority of 876 did not contain (31,4-Gal
subterminal to Fuc.
Further, 892 was not digested, indicating that it did not contain non-reducing
terminal (31,4-Gal.
When the a1,3/4 fucosidase, a1,2 fucosidase, and 81,4-galactosidase reaction
product was further
digested with fi1,3-galactosidase, the signal intensity of 892 was reduced,
indicating that it included
glycans with terminal (31,3-Gal. The signal intensity of 568 was increased
relative to 730, indicating
that also 730 included glycans with terminal P1,3-Gal.
The experimental structures of the major hESC glycosphingolipid neutral glycan
signals were thus
determined ('>' indicates the order of preference among the lipid glycan
structures of hESC;
indicates that the oligosaccharide sequence in brackets may be either branched
or unbranched;
indicates a branch in the structure):
730 Hex3HexNAci > HexiHexNAclLac > Gal(34GlcNAcLac
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876 Hex3HexNAcidHexi > Fuca2[HexiHecNAci]Lac > Fuca2Ga1(34G1cNAcLac
> Fuca3/4[HexlHecNAci]Lac
568 HexzHexNAci > HecNAcLac
933 Hex3HexNAc2 > [HexlHecNAc.z]Lac
892 Hex4HexNAci > [HexzHecNAci]Lac > Gal(33[HexiHecNAci]Lac
1095 Hex4HexNAc2 > [HexzHecNAc2]Lac > Gal(33HexNAc[Hex1HecNAci ]Lac
> Gal(34G1cNAc[HexiHecNAc1 ]Lac
1460 Hex5HexNAc3 > [Hex3HecNAc3]Lac
> Gal(34G1cNAc(Gal(34GlcNAc)[Hex1HecNAci ]Lac
Acidic lipid glycans. The mass spectrometric profile of the hESC
glycosphingolipid sialylated
glycan fraction was analyzed (not shown). The four major glycan signals,
together comprising more
than 96% of the total glycan signal intensity, corresponded to monosaccharide
compositions
NeuAciHex3HexNAci (997), NeuAclHexzHexNAci (835), NeuAciHex4HexNAci (1159),
and
NeuAc2Hex3HexNAci (1288).
The acidic glycan fraction was subjected to a2,3-sialidase digestion and the
resulting neutral and
acidic glycan fractions were purified and analyzed separately. In the acidic
fraction, signals 1159
and 1288 were digested and 835 was partly digested. In the neutral fraction,
signals 730 and 892
were the major appeared signals. These results indicated that: 1159 consisted
mainly of glycans
with a2,3-NeuAc, 1288 contained at least one a2,3-NeuAc, a major proportion of
glycans in 835
contained a2,3-NeuAc, and in the original sample a major proportion of
NeuAc1_2Hex3HexNAc1
contained solely a2,3-linked NeuAc.
EXAMPLE 6. Endo-(3-galactosidase analysis of cellular glycan types.
Endo-(3-galactosidase reaction conditions
The substrate glycans were dried in 0.5 ml reaction tubes. The endo-(3-
galactosidase (E. freundii,
Seikagaku Corporation, cat no 100455, 2.5 mU/reaction) reactions were carried
out in 50 mM Na-
acetate buffer, pH 5.5 at 37 C for 20 hours. After the incubation the
reactions mixtures were boiled
for 3 minutes to stop the reactions. The substrate glycans were purified using
chromatographic
methods according to the present invention, and analyzed with MALDI-TOF mass
spectrometry as
described in the preceding Examples.
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In similar reaction conditions with with 2 nmol of each defined
oligosaccharide control, the reaction
produced signal at m/z 568 (HexzHexNAci) as the major reaction product from
lacto-N-neotetraose
and para-lacto-N-neohexaose, but not from lacto-N-neohexaose or para-lacto-N-
neohexaose
monofucosylated at the 3-position of the inner G1cNAc residue; and sialylated
signal corresponding
to NeuAc,HexzHexNAc, from a3'-sialyl-lacto-N-neotetraose. These results
confirmed the reported
specificities for the enzyme in the employed reaction conditions.
Results with cellular glycan types
hESC O-glycans. In neutral reducing 0-glycans isolated from hESC, major
digestion products
were signals at m/z 568 (HexzHexNAci) and 714 (HexzHexNAcidHexl),
corresponding to non-
fucosylated and fucosylated non-reducing glycan fragments from poly-N-
acetyllactosamine
sequences (poly-LacNAc); and at m/z 609 (HexlHexNAc2) corresponding to another
type of glycan
fragment, including reducing end 0-glycan fragment such as Core 2
trisaccharide
Gal(33(G1cNAc(36)Ga1NAc.
Major digested glycan signals corresponding to O-glycan structures were at m/z
1136
(Hex3HexNAc3), 974 (Hex2HexNAc3), 1120 (Hex2HexNAc3dHexi), and 1282
(Hex3HexNAc3dHexi). Signal 1136 corresponded to a glycan also sensitive to
(31,3-galactosidase
exoglycosidase digestion, and therefore was determined to contain a non-
reducing end
Gal(33GlcNAc(33Ga1(34GlcNAc(3 sequence; signal 1282 corresponds to a
fucosylated derivative
thereof. Signals 974 and 1120 are non-fucosylated and fucosylated forms of 0-
glycans with non-
reducing terminal HexNAc.
hESC glycosphingolipid glycans. The major digestion product in hESC neutral
glycosphingolipid
glycans were the signals at m/z 568 (Hex2HexNAc1) and 714 (HexzHexNAcidHexi)
indicating the
presence of non-fucosylated and fucosylated poly-LacNAc sequences. Further,
the signals at m/z
1428 (Hex3HexNAc3dHex2) and 1282 (Hex3HexNAc3dHex1) were products, indicating
the presence
of different glycan terminal sequences with non-reducing terminal HexNAc than
in the
abovementioned cell types. Major sensitive signals were signals at m/z 730,
876, 933, 1095, and
1241 with similar interpretation as with CB MNC above.
In conclusion, the profiles of endo+-galactosidase reaction products
efficiently reflected cell type
specific glycosylation features as described in the preceding Examples and
they represent an
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alternative and complementary method for analysis of cellular glycan types.
Further, the present
results demonstrated the presence of linear, branched, and fucosylated poly-
LacNAc in all studied
cell types and in different glycan types including N- and 0-glycans and
glycosphingolipid glycans;
and further quantitative and cell-type specific proportions of these in each
cell type, which are
characteristic to each cell type.
hESC N-glycans. Combination of NMR spectroscopy and (31,4-galactosidase, (3-N-
acetylglucosaminidase, and (3-hexosaminidase digestions indicates that hESC
neutral
monoantennary and biantennary-size N-glycans preferentially contained LacNAc
(LN) antennae,
more preferentially in a complex-type biantennary N-glycan backbone with (31,2-
branches. Here it
was studied by endo-(3-galactosidase digestion of the hESC acidic N-glycan
fraction, which N-
glycan backbones contained poly-N-acetyllactosamine (poly-LN) antennae. The
glycan sample was
produced as described in the other Examples of the present invention from
similar hESC samples.
Biantennary N-glycan fragments that were produced with endo-(3-galactosidase
included S 1 H4N4
(m/z 1917), preferentially produced from a biantennary N-glycan with one poly-
LN antenna and
one sialylated LN antenna. According to the present invention this glycan
included an antenna
structure R-G1cNAc(33Ga1(34G1cNAc(32Man, wherein R is non-reducing N-glycan
antenna structure
according to the invention. In a further embodiment of the present invention,
the other antenna in
the same N-glycan is sialylated LacNAc, more preferably NeuAc-Gal-
G1cNAc(32Man.
EXAMPLE 7. The glycome of human embryonic stem cells reflects their
differentiation stage.
SUMMARY
Complex carbohydrate structures, glycans, are elementary components of
glycoproteins,
glycolipids, and proteoglycans. These glycoconjugates form a layer of glycans
that covers all
human cell surfaces and forms the first line of contact towards the cell's
environment. Glycan
structures called stage specific embryonic antigens (SSEA) are used to assess
the undifferentiated
stage of embryonic stem cells. However, the whole spectrum of stem cell glycan
structures has
remained unknown, largely due to lack of suitable analysis technology. We
describe the first global
study of glycoprotein glycans of human embryonic stem cells, embryoid bodies,
and further
differentiated cells by MALDI-TOF mass spectrometric profiling. The analysis
reveals how certain
asparagine-linked glycan structures characteristic to stem cells are lost
during differentiation while
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new structures emerge in the differentiated cells. The results indicate that
human embryonic stem
cells have a unique glycome and that their differentiation stage can be
identified by glycome
analysis. We suggest that knowledge about stem cell specific glycan structures
can be used for e.g.
purification, manipulation, and quality control of stem cells.
MATERIALS & METHODS
Human embryonic stem cell lines. Five Finnish hESC lines, FES 21, FES 22, FES
29, FES 30
(Skottman et al., 2005. Stem cells 23:1343-56) and FES 61 were used in the
present study. These
lines are included in the International Stem Cell Initiative (Andrews et al.,
2005. Nat. Biotechnol.
23:795-7). The cells were propagated on human foreskin fibroblast (hFF) feeder
cells in serum-free
medium (KnockoutTM, Gibco/Invitrogen). In FACS analyses 70-90% of cells from
mechanically
isolated colonies were typically Tra 1-60 and Tra 1-81 positive (not shown).
Cells differentiated
into embryoid bodies (EB, stage 2 differentiated) and further differentiated
cells grown out of the
EB as monolayers (stage 3 differentiated) were used for comparison against
hESC. The
differentiation protocol favors the development of neuroepithelial cells while
not directing the
differentiation into distinct terminally differentiated cell types (Okabe et
al., 1996. Mech. Dev.
59:89-102). EB derived from FES 30 had less differentiated cell types than the
other three EB.
Stage 3 cultures consisted of a heterogenous population of cells dominated by
fibroblastoid and
neuronal morphologies. For the glycome studies the cells were collected
mechanically, washed, and
stored frozen until analysis.
In a preferred embodiment the invention is directed to the use of data
obtained embryoid bodies or
ESC-cell line cultivated under conditions favouring neuroepithelial cells for
search of specific
structures indicating neuroepithelial development, preferably by comparing the
material with cell
materials comprising neuronal and/or epithelial type cells.
Asparagine-linked glycome profiling. Total asparagine-linked glycan (N-glycan)
pool was
enzymatically isolated from about 100 000 cells. The total N-glycan pool
(picomole quantities) was
purified with microscale solid-phase extraction and divided into neutral and
sialylated N-glycan
fractions. The N-glycan fractions were analyzed by MALDI-TOF mass spectrometry
either in
positive ion mode for neutral N-glycans or in negative ion mode for sialylated
glycans (Saarinen et
al., 1999, Eur. J. Biochem. 259, 829-840). Over one hundred N-glycan signals
were detected from
each cell type revealing the surprising complexity of hESC glycosylation. The
relative abundances
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of the observed glycan signals were determined based on relative signal
intensities (Harvey, 1993.
Rapid Commun. Mass SpectNom. 7:614-9; Papac et al., 1996. Anal. Chem. 68:3215-
23).
RESULTS
In the present study, we analyzed the N-glycome profiles of hESC, EB, and st.3
differentiated cells
(Fig. 17).
The similarity of the N-glycan profiles within the group of four hESC lines
suggested that the
obtained N-glycan profiles are a description of the characteristic N-glycome
of hESC. Overall, 10%
of the 100 most abundant N-glycan signals present in hESC disappeared in st.3
differentiated cells,
and 16% of the most abundant signals in st.3 differentiated cells were not
present in hESC. This
indicates that differentiation induced the appearance of new N-glycan types
while earlier glycan
types disappeared. In quantitative terms, the differences between the glycan
profiles of hESC, EB,
and st.3 differentiated cells were: hESC vs. EB 19%, hESC vs. st.3 24%, and EB
vs. st.3 12%.
The glycome profile data was used to design glycan-specific labeling reagents
for hESC. The
most interesting glycan types were chosen to study their expression profiles
by lectin histochemistry
as exemplified in Figure 18 for the lectins that recognize either a2,3-
sialylated (MAA-lectin, Fig.
18A.) binding to the hESC cells or a-mannosylated glycans (PSA-lectin, Fig.
18B.) binding to the
surfaces of feeder cells (MEF). The binding of the lectin reagents was
inhibited by specific
carbohydrate inhibitors, sialyla2-lactose and mannose, respectively (Fig. 18C.
and 18D.). The
results are summarized in Table 43.
Table 43 further represent differential recognition feeder and stem cells by
two other lectins,
Ricinus communis agglutinin (RCA, ricin lectin), known to recognize especially
terminal Gal(3-
structures, especially Gal04G1c(NAc)-type structures and peanut agglutinin
(PNA) reconnizing
GaUGa1NAc structures. The cell surface expression of ligand for two other
lectin RCA and PNA on
hESC cells, but only RCA ligands of feeder cells.
The present results indicate and the invention is directed to the hESC glycans
are potential targets
for recognition by stem cell specific reagents. The invention is further
directed to methods of
specific recognition and/or separation of hESC and differentiated cells such
as feeder cells by
glycan structure specific reagents such as lectins. Human embryonic stem cells
have a unique
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glycome that reflects their differentiation stage. The invention is
specifically directed to analysis of
cells according to the invention with regard to differentiation stage.
The results were also used to generate an algorithm for identification of hESC
differentiation stage
(Fig. 5). To test whether the obtained N-glycan profiles could be used for
reliable identification of
hESC and differentiated cells even with the presence of sample-to-sample
variation, a
discrimination analysis was performed on the data. The hESC line FES 29 and
embryoid bodies
derived from it (EB 29) were selected as the training group for the
calculation that effectively
discriminated the two samples (Fig. 5):
glycan score = a - b - c,
wherein a is the sum of the relative abundances (%) of all signals with
proposed compositions with
two or more dHex (F>2) in the sialylated N-glycan fraction, b is the sum of
the relative abundances
(%) of all signals with hybrid-type structures (ST= H), and c is the sum of
the relative abundances
(%) of all signals with proposed compositions with five or more HexNAc and
equal amounts of Hex
and HexNAc (H=N>5); see Table 43 for structure codes and Fig. 17 for the
dataset.
The resulting equation was applied to the other samples that served as the
test group in the analysis
and the results are described graphically in Fig. 5. hESC and the
differentiated cell samples were
clearly discriminated from each other (p < 0.01, Student's t test).
Furthermore, the st.3
differentiated cell samples were separated from the EB samples (p < 0.05, Mann-
Whitney test). The
predicted 95% confidence intervals (assuming normal distribution of glycan
scores within each cell
type) are shown for the three cell types, indicating that a calculated glycan
score has potential to
discriminate all three cell types. At 96% confidence interval, hESC and the
differentiated cell types
(EB and st.3) were still discriminated from each other (not shown in the
figure). The results indicate
that glycome profiling is a tool for monitoring the differentiation status of
stem cells.
CONCLUSIONS
The present data represent the glycome profiling of hESC:
0 hESC have a unique N-glycome comprising of over 100 glycan components
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= Differentiation induces a major change in the N-glycome and the cell surface
molecular
landscape of hESC
Utility of hESC glycome data:
= Identification of new stem cell markers for e.g. antibody development
= Quality control of stem cell products
= Identification of hESC differentiation stage
= Control of variation between hESC lines
= Effect of external factors and culture conditions on hESC status
Especially preferred uses of the data are
Use of the hESC glycome for identification of specific cell surface markers
characteristic for the
pluripotent hESCs.
The invention is directed to further analysis and production of present and
analogous glycome data
and use of the methods for further indentification of novel stem cell specific
glycosylation features
and form the basis for studies of hESC glycobiology and its eventual
applications according to the
invention
EXAMPLE 8. Identification of specific glycosylation signatures from glycan
profiles in
various steps of human embryonic stem cell differentiation.
To identify differentiation stage specific N-glycan signals in sialylated N-
glycan profiles of hESC,
EB, and stage 3 differentiated cells (see Examples above), major signals
specific to either the
undifferentiated (Fig. 19) or differentiated cells (Fig. 20) were selected
based on their relative
abundances in the database of the four hESC lines, and the four EB and st.3
cell samples derived
from the four hESC lines, respectively. The selected glycan signal groups,
from where indifferent
glycan signals have been removed, have reduced noise or background and less
observation points,
but have the resolving power. Such selected signal groups and their patterns
in different sample
types serve as a signature for the identification of, for example, 1)
undifferentiated hESC (Fig. 19),
2) differentiated cells, preferentially their differentiation stage relative
to hESC (Fig. 20), 3)
differentiation lineage, such as the neuroectodermally enriched st.3 cells
compared to the mixed cell
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population of EB (e.g. 1799), 4) glycan signals that are specific to hESC
(e.g. 2953), 5) glycan
signals that are specific to differentiated cells (e.g. 2644), or 6) glycan
signals that have individual
i.e. cell line specific variation (e.g. 1946 in cell line FES 22, 2133 in cell
line FES 29, and 2222 in
cell line FES 30). Moreover, glycan signals can be identified that do not
change during hESC
differentiation, including major glycans that can be considered as
housekeeping glycans in hESC
and their progeny (e.g. 1257, 1419, 1581, 1743, 1905 in Fig. 17.A, and 2076 in
Fig. 17.B).
Proposed glycan compositions and structure groups for the signals are
presented in Table 43.
To further analyze the data and to find the major glycan signals associated in
given hESC
differentiation stage, two variables were calculated for the comparison of
glycan signals in the N-
glycan profile dataset described above, between two samples:
1. absolute difference A = (S2 - SI ), and
2. relative difference R = A / SI,
wherein SI and S2 are relative abundances of a given glycan signal in samples
1(the four EB
samples) and 2 (the four st.3 cell samples), respectively.
When A and R were calculated for the glycan profile datasets of the two cell
types, and the glycan
signals thereafter sorted according to the values of A and R, the most
significant differing glycan
signals between the two samples could be identified. Among the fifty most
abundant neutral N-
glycan signals in the data (Fig. 17.A), the following five signals experienced
the highest relative
change R in the transition from EB to st.3 differentiated cells in the dataset
of four EB and four st.3
cell samples: 1825 (R - 5.8, corresponding to 6.8-fold increase), 1136 (R -
1.4, corresponding to
2.4 fold increase), 1339 (R = 0.9, corresponding to 1.9 fold increase), 2142
(R = 0.87,
corresponding to 87% decrease), and 2174 (R = 0.56, corresponding to 56%
decrease). Four of
these signals corresponded to complex-type structures (Table 43), indicating
that the major
differing glycan structures were included in the complex-type glycan group.
However, the majority
of the other complex-type glycan signals in the dataset were not observed to
differ as significantly
between the two cell types (i.e. they did not have large values of A and/or
R), indicating that the
procedure was able to identify st.3 cell and EB associated glycan subgroups
within the whole
complex-type glycan group. The one signal corresponding to hybrid-type
structures (1136) had the
highest value of the absolute differences A among all the glycan signals in
the neutral N-glycan
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profiles (A=0.48), indicating that also this signal had significance in the
discrimination between the
EB and st.3 cell samples in the studied dataset.
EB derived from the hESC line FES 30 were different in their overall N-glycan
profiles compared
to the other three EB samples (Fig. 17) and had the differentiation-specific
glycan score value
closer to the hESC samples (Fig. 5), correlating with the property of EB 30
having less
differentiated cell types than the other three EB. This was also seen in
distinct glycan signals, e.g.
2222 in Fig. 17.B.
EXAMPLE 9. Schematic concepts of glycome change and mass spectrometric
screening.
Introduction to glycomics
All human cell types have unique glycome - an entity of all glycans of the
cell, present mainly on
cell surface glycoproteins and glycolipids, including the SSEA and Tra glycan
antigens. However,
the whole spectrum of hESC glycan structures (the stem cell glycome) is still
unknown. Glycans,
the complex carbohydrate structures, are capable of great structural variation
and their specific
molecular structures carry diverse biological information.
EXAMPLE 10.
Data preparation
The mass data was normalized by dividing selected peaks with the total sum of
the peak intensities
of the corresponding spectra. Finally, normalized mass data from hESC,
embryonic bodies, and
stage 3 differentiated hESC was tabulated in Excel spread sheet and imported
in Statistica 7.0
software (StatSoft).
Data cleaning
Neutral and acidic glycans
In certain cases sample were divided into two tubes and MALDI was performed
separately. In these
cases data from the separate shots were combined and represented by their
average intensity.
If all or almost all data values were zero, the corresponding mass was removed
from the data set.
For analyses requiring variance such as one way ANOVA and Factor analysis,
further removal of
masses were performed if all or almost all values were zeros in some
subcategory.
EXAMPLE 11
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ANOVA
One way ANOVA was performed to analyze basic statistics of the data. The
means, standard
deviations, box and whisker blots were screened to have an overall view of the
data and to identify
mass peaks with variation between different cell lines or differentiation
stage. The one way
ANOVA analysis was performed in Statistica with Fisher LSD post hoc analysis.
EXAMPLE 12
Factor analysis
Factor analysis was employed in order to find "hidden" factors which would
explain the variation
within the mass distribution and their intensities. Moreover, by using factor
analysis, the total
variation could be explained with a smaller number of variables which
simplifies the analysis.
The factor analysis (Principal component, Varimax normalised, Eigenvalues >
1.0, factor loadings
> 0.62) indicated 7 to 8 main factors when explained variance > 5% was
considered as a cut off for
a factor to be included into the model.
The 8 factors for acidic glycans comprised in the following masses:
F1 : 1678, 1727, 1873, 1889, 1914, 2002, 2367, 2441, 2732, 2807, 2880, 3099
and 3172
F2 : 1475, 1637, 1799, 2076, 2133, 2482, and 2714
F3 : 2221, 2279, 2280, 2570, 2571, 2644, 2645, 2936, and 3098
F4 : 1354, 1500, 1516, 1541, 1791, 2010, 2156, 2230, 2246, and 2447
F5 : 2011, 2321, and 2603
F6 : 2254, 2528, 2544, 3025 and 3390
F7:3024
F8: 2400 and 3170
The 7 factors for neutral glycans comprised in the following masses:
F1 : 609, 771, 892, 933, 1054, 1095, 1216, 1378, 1540, 1702, 1743, 1809, 1955,
2028 and 2174
F2 : 1460, 1485, 1606, 1622, 1647, 1704, 1850, 1866 and 2021
F3 : 917, 1120, 1241, 1282, 1298, 1339, 1403, 1444, 1501, 1793, 1987 and 1996
F4 : 1136, 1209, 1590, 2158, 2391 and 2466
F5 : 730, 1031, 1565, 1825, 2117 and 2304
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F6 : 1257 and 1905
F7 : 1784 and 2229
CORRELATION MATRIX, NEUTRAL N-GLYCAN FRACTION
Soluble HexNAc1-glycans H(4-9)N1 intercorrelate significantly. The correlation
matrix reveals
two subgroups: 1) H4N1, H5N1, and H6N1 comprising smaller soluble HexNAcl-
glycans H(4-
6)N1; and 2) H6N1, H7N1, H8N1, and H9N1 comprising larger soluble HexNAcl-
glycans H(6-
9)N1. H3N1 correlates most significantly with H4N1 but not with the other
soluble HexNAcl-
glycans.
The soluble HexNAcl -glycans further negatively correlate with low-mannose
type N-glycans, most
significantly with non-fucosylated low-mannose type N-glycans H2N2, H3N2, and
H4N2; and with
complex-type N-glycans with H=N terminal HexNAc composition feature, most
significantly with
H5N5F3.
The soluble HexNAcl-glycans further negatively correlate with complex-type N-
glycans, most
significantly with H5N4, H5N4F1, H6N5, and H6N5F1; and with high-mannose type
N-glycans,
most significantly with H8N2.
High-mannose type N-glycans H(6-8)N2 intercorrelate significantly; whereas
H9N2 correlates
significantly with glucosylated high-mannose type N-glycan H10N2; and H5N2
negatively
correlates with the larger H(6-9)N2 glycans, most significantly with H9N2; and
the fucosylated
high-mannose type N-glycans H5N2F1 and H6N2F1 correlate significantly with the
fucosylated
low-mannose type N-glycans. Therefore, the correlation matrix reveals four
differently regulated
groups within the high-mannose type N-glycans: 1) H5N2, 2) H(6-8)N2, 3) H(9-
10)N2, and 4) H(5-
6)N2F1; groups 3) and 2) are preferentially expressed in hESC; and 1) and 4)
in the differentiated
cell types.
In the following analysis of the performed factor analyses, glycan signals
were assigned into glycan
structure classes as described in the present invention and coded by the
following one letter-code:
A = acidic, C = complex-type, H = hybrid-type, S = soluble HexNAcl-type, O=
other types, L
low-mannose type, M = high-mannose type, N = monoantennary type, B =
biantennary-size
complex-type, R = larger than biantennary-size complex-type, F = fucosylated,
E = complex-
fucosylated i.e. containing more than one dHex residue, P = sulphated or
phosphorylated, T =
terminal HexNAc, wherein n(N) > n(H), Q = terminal HexNAc, wherein n(N) =
n(H), X = terminal
Hex in complex-type N-glycan, wherein n(H) > n(N) +1, A = acetylated, Y =
containing N-
glycolylneuraminic acid.
FACTOR ANALYSIS, NEUTRAL N-GLYCAN FRACTION
Factor 1 reflects positive contribution of:
1) soluble HexNAcl-type glycans, preferably including H(4-9)N1, and
2) non-fucosylated low-mannose type N-glycans, preferably including H(2-4)N2;
and negative contribution of:
3) large high-mannose type N-glycans, preferably including H(7-8)N2,
4) neutral biantennary-size complex-type N-glycans, preferably including
H5N4F(1-2),
5) neutral triantennary-size complex-type N-glycans, preferably including
H6N5F(0-1), and
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6) HIN21ow-mannose type N-glycans.
In a preferred embodiment of the present invention, Factor 1 reflects a switch
between glycan
groups Factor 1-1 and Factor 1-2; and glycan groups Factor 1-3, Factor 1-4,
Factor 1-5, and Factor
1-6. In a further preferred embodiment, relative high expression of one or
more of the first glycan
groups is associated with relative low expression of the latter glycan groups,
and vice versa. In
another further preferred embodiment, the first glycan groups are associated
with differentiated
cells and the latter glycan groups are associated with hESC.
Positive contribution:
H6N1 S 1216 0,86
H7N1 S 1378 0,86
H9N1 S 1702 0,85
H8N1 S 1540 0,82
H4N1 S 892 0,81
H3N2 L 933 0,81
H4N2 L 1095 0,78
H5N1 S 1054 0,78
H2N2 L 771 0,72
Negative contribution:
H5N4F2 C B E 1955 -0,83
H1N2 L 609 -0,79
H6N5F1 C R F 2174 -0,79
H5N4F1 C B F 1809 -0,78
H8N2 M 1743 -0,74
H6N5 C R 2028 -0,73
H7N2 M 1581 -0,66
Factor 2 reflects negative contribution of:
1) neutral complex-type N-glycans with N>H type non-reducing terminal HexNAc,
preferably
including H4N5, H4N5F3, or H3N4F1,
2) neutral complex-type N-glycans with N=H type non-reducing terminal HexNAc,
preferably
including H5N5(FO-1) or H4N4F1, and
3) neutral large hybrid-type N-glycans, preferably including H5N3(FO-1) or
H6N3.
In a preferred embodiment of the present invention, Factor 2 reflects the
relative amount of the
glycan groups Factor 2-1, Factor 2-2, or Factor 2-3. In a further preferred
embodiment, these glycan
groups are associated with differentiated cells.
Negative contribution:
H4N4F1 C F Q 1647 -0,67
H5N5F1 C F Q 2012 -0,71
H5N3 H 1460 -0,77
H6N3 H 1622 -0,79
H3N4F1 C F T 1485 -0,80
H5N3F1 H F 1606 -0,81
H5N5 C Q 1866 -0,86
H4N5 C T 1704 -0,88
H4N5F3 C E T 1850 -0,90
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Factor 3 reflects positive contribution of:
1) neutral small hybrid-type or monoantennary N-glycans, preferably including
H4N3;
and negative contribution of:
2) neutral fucosylated monoantennary N-glycans, preferably including H(2-
3)N2F1,
3) fucosylated low- and high-mannose type N-glycans, preferably including H(4-
5)N2F 1,
4) neutral complex-type N-glycans with N>H type non-reducing terminal HexNAc,
preferably
including H3N4 or H4N5F2, and
5) neutral complex-type N-glycans with N=H type non-reducing terminal HexNAc,
preferably
including H4N4 or H4N4F2.
In a preferred embodiment of the present invention, Factor 3 reflects a switch
between glycan
groups Factor 3-1 and glycan groups Factor 3-2, Factor 3-3, Factor 3-4, and
Factor 3-5. In a further
preferred embodiment, relative high expression of the first glycan group is
associated with relative
low expression of the latter glycan groups, and vice versa. In another further
preferred embodiment,
the first glycan group is associated with hESC and the latter glycan groups
are associated with
differentiated cells.
Positive contribution:
H4N3 H 1298 0,78
Negative contribution:
H4N2F1 L F 1241 -0,71
H4N4F2 C E Q 1793 -0,72
H3N4 C T 1339 -0,81
H5N2F1 M F 1403 -0,81
H4N4 C Q 1501 -0,82
H4N5F2 C E T 1996 -0,86
H2N3F1 H N F T 1120 -0,88
H3N3F1 H N F 1282 -0,91
Factor 4 reflects positive contribution of:
1) neutral monoantennary or small hybrid-type N-glycans, preferably including
H3N3 or
H4N3F2, and
2) neutral complex-type N-glycans with N=H type non-reducing terminal HexNAc
and
complex fucosylation, preferably including H5N5F2.
In a preferred embodiment of the present invention, Factor 4 reflects the
relative amount of the
glycan groups Factor 4-1 and Factor 4-2. In a further preferred embodiment,
these glycan groups
are associated with differentiated cells.
Positive contribution:
H5N5F2 C E Q 2158 0,82
H3N3 H N 1136 0,82
H4N3F2 H E 1590 0,67
Factor 5 reflects positive contribution of:
1) small soluble HexNAcl-type glycans, preferably including H3N1,
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2) neutral complex-type N-glycans with N=H type non-reducing terminal HexNAc
and
complex fucosylation, preferably including H5N5F3, and
3) fucosylated high-mannose type N-glycans, preferably including H6N2F1.
In a preferred embodiment of the present invention, Factor 5 reflects the
relative amount of the
glycan groups Factor 5-1, Factor 5-2, and Factor 5-3. In a further preferred
embodiment, these
glycan groups are associated with differentiated cells.
Positive contribution:
H5N5F3 C E Q 2304 0,85
H6N2F1 M F 1565 0,79
H3N1 S 730 0,77
Factor 6 essentially reflects the positive contribution of small high-mannose
type N-glycans (Factor
6-1), preferentially including H5N2 (positive contribution: 0.69), and
negative contribution of large
high-mannose type N-glycans (Factor 6-2), preferentially including H9N2
(positive contribution: -
0.80). In a preferred embodiment of the present invention, Factor 6 reflects a
switch between these
glycan groups, wherein relative increase in one group is reflected in relative
decrease in the other
group. In a further preferred embodiment, Factor 6-1 is associated with
differentiated cells and
Factor 6-2 is associated with hESC.
FACTOR ANALYSIS, ACIDIC AND NEUTRAL N-GLYCAN FRACTIONS
Factors Al and A2 are mainly composed of contribution of neutral glycan
signals.
Factor A3 reflects positive contribution of:
1) sialylated complex-type N-glycans with N>H type non-reducing terminal
HexNAc,
preferably including SIH4N5F(1-2),
2) sialylatedmonoantennary-type N-glycans, preferably including SIH4N3F1, and
3) large high-mannose type N-glycans preferably including H6N2;
and negative contribution of:
4) sulphated or phosphorylated N-glycans, preferably including H3N4FIP1, S(0-
2)H5N4FIP1,
S(0-1)H5N4P1, H4N3P1, S1H4N3F1P1, H4N4P1, S1H5N4F3P1, H6N5F1P1, and
H6N5F3P1; wherein P is preferably sulphate ester.
In a preferred embodiment of the present invention, Factor A3 reflects a
switch between glycan
groups Factor A3-1, Factor A3-2, and Factor A3-3; and glycan group Factor A3-
4. In a further
preferred embodiment, relative high expression of the first glycan group is
associated with relative
low expression of the latter glycan groups, and vice versa. In another further
preferred embodiment,
the first glycan group is associated with hESC and the latter glycan groups
are associated with
differentiated cells.
Positive contribution:
H6N2 M 1419 0,87
S1H4N5F1 A S1 C F T 2117 0,71
S1H4N3F1 A S1 H N F 1711 0,65
S1H4N5F2 A S1 C E T 2263 0,60
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Negative contribution:
H3N4F1P1 A C F P T 1541 -0,89
S1H5N4F1P1 A S1 C B F P 2156 -0,88
H5N4F1P1 A C B F P 1865 -0,86
S1H5N4P1 A S1 C B P 2010 -0,83
H4N3P1 A H P 1354 -0,83
S1H4N3F1P1 A S1 H N F P 1791 -0,78
H4N4P1 A C P Q 1557 -0,74
S2H5N4F1P1 A S2 C B F P 2447 -0,72
S1H5N4F3P1 A S1 C B E P 2448 -0,71
H6N5F1P1 A C R F P 2230 -0,70
H5N4P1 A C B P 1719 -0,66
H6N5F3P1 A C R E P 2522 -0,66
Factor A4 reflects positive contribution of:
1) sialylated and neutral complex-type biantennary-size N-glycans, preferably
including
S1H5N4F(0-1) and H5N4F1;
and negative contribution of:
2) small disialylated glycans, preferably including S2H(2-4)N2F1 and S2H(2-
3)N3F 1,
3) sialylated and neutral complex-type N-glycans with N=H type non-reducing
terminal
HexNAc, preferably including H5N5F3, SIH5N5, and H5N5FIP1,
4) fucosylated high-mannose type N-glycans, preferably including H6N2F 1, and
5) sialylated and neutral complex-type N-glycans with N>H type non-reducing
terminal
HexNAc, preferably including S1H5N6F2 and H3N5F1.
In a preferred embodiment of the present invention, Factor A4 reflects a
switch between glycan
group Factor A4-1; and glycan groups Factor A4-2, Factor A4-3, Factor A4-4,
and Factor A4-5. In
a further preferred embodiment, relative high expression of the first glycan
group is associated with
relative low expression of the latter glycan groups, and vice versa. In
another further preferred
embodiment, the first glycan group is associated with hESC and the latter
glycan groups are
associated with differentiated cells.
Positive contribution:
S1H5N4F1 A S1 C B F 2076 0,67
S1H5N4 A S1 C B 1930 0,63
G1H5N4F1 A S1 C B F Y 2092 0,56
H5N4F1 C B F 1809 0,50
Negative contribution:
S2H3N2F1 A S2 0 F 1637 -0,90
H5N5F3 C E Q 2304 -0,89
S2H2N2F1 A S2 0 F 1475 -0,87
S2H4N2F1 A S2 0 F 1799 -0,85
H6N2F1 M F 1565 -0,77
S1H5N6F2 A S1 C E T 2482 -0,76
H3N5F1 C F T 1688 -0,74
H5N5F1P1 A C F P Q 2068 -0,73
S1H5N5 A S1 C Q 2133 -0,69
S2H2N3F1 A S2 0 F 1678 -0,61
S2H3N3F1 A S2 H N F 1840 -0,57
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Factor A5 reflects negative contribution of:
1) neutral fucosylated monoantennary or hybrid-type N-glycans, preferably
including H(2-
4)N3F1,
2) fucosylated low- and high-mannose type N-glycans, preferably including H(4-
5)N2F 1,
3) neutral complex-type N-glycans with N>H type non-reducing terminal HexNAc,
preferably
including H4N5F2 and H3N4, and
4) neutral complex-type N-glycans with N=H type non-reducing terminal HexNAc,
preferably
including S1H5N5F1A1, H4N4F2, and H4N4.
In a preferred embodiment of the present invention, Factor A5 reflects a
switch in relative amounts
of glycan groups Factor A5-1, Factor A5-2, Factor A5-3, and Factor A5-4. In a
further preferred
embodiment, these glycan groups are associated with differentiated cells.
Negative contribution:
H2N3F1 H N F T 1120 -0,85
S1H7N5F1 A S1 C F X 2603 -0,82
H4N2F1 L F 1241 -0,80
H5N2F1 M F 1403 -0,79
H3N3F1 H N F 1282 -0,78
H2N4F1 0 F T 1323 -0,76
H4N5F2 C E T 1996 -0,75
S1H5N5F1A1 A S1 C F Q A 2321 -0,75
H3N4 C T 1339 -0,74
H4N4F2 C E Q 1793 -0,73
H4N3F1 H F 1444 -0,71
H4N4 C Q 1501 -0,70
Factor A7 reflects positive contribution of:
1) sialylated hybrid-type N-glycans, preferably including S1H5N3F(0-1) and
H6N3,
2) small disialylated glycans, preferably including S2H2N3F1 and S2H4N3F1,
3) small high-mannose type N-glycans, preferably including H5N2,
and negative contribution of:
4) large monosialylated complex-type N-glycans, preferably including S1H7N6F1,
S(1-
2)H6N5F1, S1H8N7F1, and S1H7N6F3, and
5) large high-mannose type and glucosylated N-glycans, preferably including
H9N2 and H(10-
11)N2.
In a preferred embodiment of the present invention, Factor A7 reflects a
switch between glycan
groups Factor A7- 1, Factor A7-2, and Factor A7-3; and glycan groups Factor A7-
4 and Factor A7-
5. In a further preferred embodiment, relative high expression of the first
glycan group is associated
with relative low expression of the latter glycan groups, and vice versa. In
another further preferred
embodiment, the first glycan group is associated with differentiated cells and
the latter glycan
groups are associated with hESC.
Positive contribution:
S1H6N3 A Sl H 1889 0,89
S1H5N3F1 A S1 H F 1873 0,80
S1H5N3 A S1 H 1727 0,72
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S2H2N3F1 A S2 0 F 1678 0,70
S2H4N3F1 A S2 H N F 2002 0,64
H5N2 M 1257 0,58
Negative contribution:
S1H7N6F1 A S1 C R F 2807 -0,75
S1H6N5F1 A S1 C R F 2441 -0,71
S1H8N7F1 A S1 C R F 3172 -0,70
S1H7N6F3 A S1 C R E 3099 -0,68
H1ON2 M G 2067 -0,64
S2H6N5F1 A S2 C R F 2732 -0,62
H9N2 M 1905 -0,55
H 11 N2 M G 2229 -0,52
Factor A8 reflects positive contribution of:
1) complex-fucosylated complex-typeN-glycans, preferably including S1H6N5F2
and
S1H5N4F(2-3);
and negative contribution of:
2) multisialylated biantennary-size complex-type N-glycans, preferably
including S2H5N4 and
S2H5N5F1,
3) sialylated complex-type N-glycans with N=H type non-reducing terminal
HexNAc,
preferably including S(1-2)H6N6F1 and S(1-2)H5N5F1, and
4) 0-acetylated sialylated N-glycans, preferably including G1H6N5F2A1 and
G1H5N4F2A1,
or S1H7N5F1A1 and S1H6N4F1A1.
In a preferred embodiment of the present invention, Factor A8 reflects a
switch between glycan
group Factor A8-1; and glycan groups Factor A8-2, Factor A8-3, and Factor A8-
4. In a further
preferred embodiment, relative high expression of one or more of the first
glycan groups is
associated with relative low expression of the latter glycan groups, and vice
versa. In another
further preferred embodiment, the first glycan group is associated with hESC
and the latter glycan
groups are associated with differentiated cells.
In a further preferred embodiment of the present invention, Factor A8 reflects
a switch between N-
glycan antenna sialylation (Factor A8-2) and fucosylation (Factor A8-1).
Positive contribution:
S1H6N5F2 A S1 C R E 2587 0,65
G1H5N4F2 A S1 C B E Y 2238 0,60
S1H5N4F2 A S1 C B E 2222 0,60
S1H5N4F3 A S1 C B E 2368 0,57
Negative contribution:
G1H6N5F2A1 A S1 C E AY 2645 -0,90
S2H6N6F1 A S2 C R F Q 2936 -0,87
S2H7N6F1 A S2 C R F 3098 -0,87
S1H6N6F1 A S1 C R F Q 2644 -0,86
S2H5N4 A S2 C B 2221 -0,84
H7N3 H 1784 -0,80
S2H5N5F1 A S2 C F Q 2570 -0,77
S1H5N5F1 A S1 C F Q 2279 -0,76
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S1H5N5F3 A S1 C E Q 2571 -0,69
G1H5N4F2A1 A S1 C E AY 2280 -0,60
The results of this analysis are gathered in Tables 50 and 51 for hESC-
associated and differentiated
cell-associated identified glycan structure groups, respectively.
EXAMPLE 13
Correlation analysis
Pearson Correlation analysis was performed in Statistica and correlations >
0.7 or < -0.7 were
considered relevant (see Tables 30 and 31).
EXAMPLE 14
Discriminant function analysis of neutral N-glycans
The statistically significant mass intensities (p<0.099) shown in Table 25
were used in Forward
Stepwise Discriminant Analysis. The tolerance was 0.010, F value of 1.0 was
used instead of the
default value one in order to inerease the statistical significance of the
model.
Results
The Partial Wilks' Lambda in Table 32 indicates variables - in decreasing
order of contribution - to
the overall discrimination of the model. As highlighted below, the mass `2028'
is the most
significant followed by 1825, 1054, 1419, 1688, 1905, 1095, 892, 1393 and mass
`1540' contributes
the least to the overall discrimination. As the discrimination of the present
model appeared to be
high as shown in Root 1 and Root 2 (Figure 28) and Eigenvalue of the Root 1
(543.7) compared to
Root 2 (19.0) we performed removal of one mass by mass to limit the minimum
number of masses
to be able to discriminate undifferentiated human embryonic stem cells from
embryoid bodies and
stage 3 cells.
From Table 33 we notice that all p-levels are less than 0.05 meaning that all
are significant.
Furthermore this indicates that all centroids are well apart, i.e. the model
discriminates very well
between groups.
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Canonical analysis Chi-squared test identified two statistically significant
functions (canonical
roots) which discriminate between hESC, EB and st3 and also to what percentage
degree they
discriminate.
From Table 34 we conclude that 543.7/(543.7+19.0) = 96.7 % of all
discriminatory power is
explained by first function, whereas the second function only explains
19.0/(543.7+19.0) = 3.3 %.
From Table 35 we identify the coefficients for each of the independent
variables. The first
discriminant function is weighted most heavily by the masses 1393, 1688 and
1540.
From Table 36, we identify the means of canonical variables. In this case we
notice that the first
discriminant function (Root 1) discriminates mostly between EB and st3.
The second discriminant function seems to distinguish mostly between hESC and
EB/st3; however
the magnitude of the discrimination is much smaller (3.3%).
In Figure 28 this is represented more clearly. Root 1 is represented on the x-
axis and Root 2 on the
y-axis. From the figure we can see that the means are further differentiated
on the x-axis and
therefore we use Root 1 to determine the function.
Search for minimal discriminant model
The original 10 masses identified from the first discrimination analysis was
further subjected to one
by one mass removal to identify the minimum masses still able to discriminate
between groups.
This was done by removing the smallest Partial Wilks' Lambda and performing
above identified
analysis. The second minimal set of masses to be able to discriminate
comprises 5 masses shown in
Table 37.
From Table 38 we conclude that 5.7/(5.7+1.8) = 76 % of all discriminatory
power is explained by
first function, whereas the second function explains 1.8/(5.7+1.8) = 24 %.
From Table 38 it can be
noticed that all p-levels are less than 0.05 meaning that all are significant.
Furthermore this
indicates that all centroids are well apart, i.e. the model discriminates very
well between groups.
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Model Function(s)
Based on the above raw coefficients the following models can be presented:
First function (10 masses)
Y = 7.58*2028 - 87.72*1393 - 20.37*1825 - 1.61*1419 + 26.91*1688 - 23.81*1540
+ 2.47*1905 +
22.11*892 - 19.17*1095 - 3.66*1054 + 35.85
Y = differentiation degree
Second minimal function (5 masses)
Y = - 2.97*892 + 4.94*1540 - 1.03*1905 + 16.50*1393 - 11.73*1688 + 15.56
First minimal function (4 masses)
Y = 2.72*892 - 3.36*1540 + 0.64*1905 + 3.31*1688 - 10.62
EXAMPLE 15
Factor analysis for neutral and acidic glycans
Factor analysis was performed for combined data set for neutral and acidic
glycans as described
above. 8 factors were found which had explained more than 5% of total variance
(Table 39).
EXAMPLE 16
Discriminant analysis for acidic glycans
Discriminant analysis was performed as described above using Statistica
General Discriminant
Analysis module with the following parameters
Parameters: F to enter = 5 and remove = 2.0, and tolerance = 0.0 10
EXAMPLE 17
Discriminant analysis for neutral and acidic glycans
Discriminant analysis was performed as described above using Statistica
General Discriminant
Analysis module with the following parameters
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Parameters: F to enter and remove = 1.0
p-value >0.05
Example 18
FACS and immunohistochemical analysis of embryonic stem cells
Immunohistochemical staining of stem cells.Immunohistochemical studies of
embryonic stem
cells (in culture)(GF series of stainings). hESC were cultured as described in
the Examples, fixed
and after rinsing with PBS the stem cell cultures/sections were incubated in
3% highly purified
BSA in PBS for 30 minutes at RT to block nonspecific binding sites. Primary
antibodies (GF279,
288, 287, 284, 285, 283,286,290 and 289) were diluted (1:10) in PBS containing
1% BSA-PBS and
incubated lhour at RT. Other antibodies indicated in the Tables were used
similarily. After rinsing
three times with PBS, the sections were incubated with biotinylated rabbit
anti-mouse, secondary
antibody (Zymed Laboratories, San Francisco, CA, USA) in PBS for 30 minutes at
RT, rinsed in
PBS and incubated with peroxidase conjugated streptavidin (Zymed Laboratories)
diluted in PBS.
The sections were finally developed with AEC substrate (3-amino-9-ethyl
carbazole; Lab Vision
Corporation, Fremont, CA, USA). After rinsing with water counterstaining was
performed with
Mayer's hemalum solution.
Antibodies, their antigens/epitopes and codes used in the immunostainings.
Table 19 shows
antibody binding to purified glycosphingolipid fractions from small amounts of
cells
(corresponding to hundreds of thousands of cells). The binding was analysed by
TLC overlay assay
using radiolabelled antibodies. The positive signals indicate presence of
substantial amounts of the
glycolipids and minus no signal due to too low amount for analysis..
Flow cytometry. Flow cytometric analysis of lectin binding was used to study
the cell surface
carbohydrate expression of hESC. The cells were washed with PBS. The cells
were harvested into
single cell suspensions by 0.02% Versene solution (pH 7.4). Detached cells
were centrifuged at
1100g for five minutes at room temperature. Cell pellet was washed twice with
1% HSA-PBS,
centrifuged at 1100g and resuspended in 1% HSA-PBS. Cells were placed in
conical tubes in
aliquots of approximately 100000 cells each. Cell aliquots were incubated with
one of the FITC
labelled lectin for 30 minutes +4C. After incubation cells were washed with 1%
HSA-PBS,
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centrifuged and resuspended in 1% HSA-PBS. Untreated cells were used as
controls. Lectin binding
was detected by flow cytometry (FACSCalibur, Becton Dickinson).
In antibody analysis primary antibodies were incubated with suitable dilution
based on
recommendation of the producer for 30 minutes at +4C and washed once with 0.3%
HSA-PBS
before secondary antibody detection with FITC secondary antibody for 30
minutes at +4C in the
dark. As a negative control cells were incubated without primary antibody and
otherwise treated
similar to labelled cells. Cells were analysed with BD FACS Calibur (Becton
Dickinson). Results
were analysed with Cell Quest Pro software (Becton Dickinson).
Fluorecently labeled lectins were from EY Laboratories (USA) or Vector
Laboratories (UK).
Antibody origin and codes are indicated in Table 20.
Results from FACS analysis
The lectin labelling results are present in Table 45 and Figure 31 and 18 from
separate experiment
for comparision. The symbol + indicates labelling majority of cell, +/-
indicates labelling of
substantial subpopulation and (+/-) indicates weak labelling or labelling of
minor cell
population/few individual cells.
The antibody labelling results are present in Tables 46-8 and Figure 32 with
comparison to
immunohistochemistry (immuno) results. The negativity - indicates negative or
low labelling of less
than 10 % of cells when labelling with the specific antibody clone (defined in
Table 20). The four
most effective binders (for antigens H type II, H type I, type I LacNAc (Lewis
c) and globotriose)
were indicated with + in FACS Tables 46-47. These antibodies are especially
preferred for
recognition of the glycans under FACS conditions.
It is further realized that part of the structures indicated to be present can
be recognized with other
antibodies specific for the correct elongated glycan epitopes (e.g. Lewis x
structures). The binding
of LTA lectin verified the structural analysis of Lewis x on the specific N-
glycan structures and the
invention is specifically directed to known regents for the recognition of the
N-glycan linked Lex
according ot the invention. The schistosoma directed LacdiNAc specific
antibodies form Leiden
university appear not to be very effective in the recognition of the preferred
N-glycan linked
LacdiNAcs.
The comparision of the immunohistochemistry and FACS results indicates that
the due to technical
reasons FACS may be as effective for recognition of glycans observable by
immunohistochemistry.
The immunohistochemistry further reveals structures present in a few cells
observable as very weak
signals in FACS.
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EXAMPLE 24. Gene expression and glycome profiling of human embryonic stem
cells.
RESULTS AND DISCUSSION
Obtaining of the gene expression data from the hESC lines FES 21, 22, 29, and
30 has been
described (Skottman et al., 2005) and the present data was produced
essentially similarily. The
results of the gene expression profiling analysis with regard to a selection
of potentially glycan-
processing and accessory enzymes are presented in Table 49, where gene
expression is both
qualitatively determined as being present (P) or absent (A) and quantitatively
measured in
comparison to embryoid bodies (EB) derived from the same cell lines.
Fucosyltransferase expression levels. Three fucosyltransferase transcripts
were detected in hESC:
FUT1 (a1,2-fucosyltransferase; increased in all FES cell lines), FUT4 (a1,3-
fucosyltransferase IV;
increased in all FES cell lines), and FUT8 (N-glycan core a1,6-
fucosyltransferase). The data
supports the analysis of the presence of the preferred fucosylated structures
in the non-differentiated
stem cells.
Hexosaminyltransferase expression levels. The following transcripts in the
selection of Table 49
were detected in hESC: MGAT3, MGAT2 (increased in three FES cell lines),
MGAT1, GNT4b,
P3G1cNAc-T5, (33G1cNAc-T7, (33G1cNAc-T4 (present in two FES cell lines),
(36G1cNAcT
(increased in one FES cell line), i(33G1cNAcT, globosideT, and a4G1cNAcT
(present in two FES
cell lines).
Other gene expression levels. The following transcripts in the selection of
Table 49 were detected
in hESC: AERI (increased in all FES cell lines), AGO61, (33GALT3, MAN1C1, and
LGALS3.
In addition to fucosyltransferases I(FUTI), IV (FUT4), and VIII (FUT8), the
expression of
fucosyltransferase II (FUT2) was also detected in the hESC samples according
to probe with the
Affymetric code 210608_s_at. The expression was detected as "present" in hESC,
but not
significantly overexpressed compared to the embryoid bodies.
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The product of the FUT2 gene is responsible for the synthesis of Fuca2Ga1
sequences, more
preferably Fuca2Ga1(33HexNAc, wherein HexNAc is either G1cNAc or GaINAc.
According to the
present invention, this gene product preferably fucosylates glycoconjugates in
hESC specifically
forming Fuca2Gal sequences (H antigens), more preferably Fuca2Ga1(33G1cNAc(3
(H type 1),
Fuca2Ga1p3Ga1NAca (H type 3), and/or Fuca2GalP3GaLNAcP (H type 4, Globo H) in
hESC
glycoconjugates including glycosphingolipid and glycoprotein glycans as
described in the present
invention.
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TABLES
Table 1. Neutral N-glycan difference analysis.
composition' m/zz class3) fold4~ composition m/z class fold
+++ hESC5) + Differentiated
H1N2 609 M 13,88 H5N3F1 1606 H 0,98
H6N5F1 2174 C 3,33 H3N4F1 1485 C 0,92
H6N5 2028 C 3,10 H5N3 1460 H 0,89
H5N4F1 1809 C 2,26 H6N3F1 1768 H 0,81
H5N4F2 1955 C 2,26 H5N2 1257 M 0,76
++ hESC H4N2 1095 M 0,73
H4N5F3 2142 C 1,61 H6N3 1622 H 0,72
H5N4F3 2101 C 1,56 H5N5F1 2012 C 0,66
+ hESC ++ Differentiated
H11N2 2229 M 1,49 H5N5 1866 C 0,65
H5N4 1663 C 1,32 H3N3 1136 H 0,59
H10N2 2067 M 1,28 H3N2 933 M 0,58
H8N2 1743 M 1,23 H3N3F1 1282 H 0,57
H9N2 1905 M 1,16 H4N2F1 1241 M 0,57
H4N3F1 1444 H 1,13 +++ Differentiated
H7N2 1581 M 1,10 H3N2F1 1079 M 0,46
H4N3 1298 H 1,08 H4N3F2 1590 H 0,42
H4N4F1 1647 C 1,08 H3N5F1 1688 C 0,31
H6N2 1419 M 1,04 H5N2F1 1403 M 0,31
H4N5 1704 C 1,02 N2N2F1 917 M 0,29
H4N5F1 1850 C 0,24
H2N4F1 1323 A 0,24
H2N2 771 M 0,24
H4N4 1501 C 0,19
H4N4F2 1793 C 0,16
H4N5F2 1996 C 0,15
H6N4 1825 C 0,13
H3N5 1542 C 0,12
H6N2F1 1565 M 0
H2N3F1 1120 H 0
H7N4 1987 C 0
Proposed composition wherein the monosaccharide symbols are: H, Hex; N,
HexNAc; F, dHex.
2) Calculated mlz for [M+Na]+ ion rounded down to next integer.
3) N-glycan class symbols are: H, hybrid-type or monoantennary; C, complex-
type; 0, other type; F,
fucosylated; E, complex-fucosylated, wherein at least one fucose residue is
a1,2-, a1,3- or a1,4-linked.
4) `fold' is calculated as the relation of glycan signal intensities in hESC
compared to differentiated cell types
~hESC and St.3); 0, not detected in hESC.
) Association with differentiation type based on fold calculation: + low
association, ++ substantial
association, +++ high association.
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Table 2. Sialylated N-glycan difference analysis.
composition') m/zz) class3) fold4~ composition m/z class fold
+++ hESC5) + Differentiated
S1H7N6F2 2953 CE oo S2H7N6F1 3098 CF 0,75
S1H8N7F1 3172 CF ~ S1H5N5F2 2425 CE 0,71
S1H7N6F3 3099 CE 15,67 S2H5N4 2221 C 0,70
S2H4N5F1 2408 CF 5,07 S1H4N3F1 1711 HF 0,69
G2H5N4 2253 C 4,56 S1H4N3 1565 H 0,68
G1H5N4 1946 C 4,50 ++ Diff
S1H5N4F2 2222 CE 3,81 S1H4N5F1 2117 CF 0,66
S2H6N4 2383 C 3,51 S2H5N3F1 2164 HF 0,56
G1H5N4F1 2092 CF 3,13 S1H5N3 1727 H 0,52
S1H6N5F2 2587 CE 2,94 +++ Diff
S1G1H5N4 2237 C 2,68 S1H6N3 1889 H 0,47
S1H6N4F2 2384 CE 2,42 S2H3N3F1 1840 OF 0,30
S1H5N4F3 2368 CE 2,02 S1H4N4F1 1914 CF 0,29
++ hESC S1H5N3F1 1873 HF 0,28
S2H5N4F1 2367 CF 1,83 S2H2N3F1 1678 OF 0,27
S3H6N5 2878 C 1,82 S2H4N3F1 2002 OF 0,20
S2H6N5F1 2732 CF 1,80 S2H5N5F1 2570 CF 0,19
S1H4N5F2 2263 CE 1,59 S1H5N5F1 2279 CF 0,17
+hESC S1 H5N5 2133 C 0,15
S2H6N5F2 2879 CE 1,49 S1H6N4F1Ac 2280 CF 0,13
S1H7N6F1 2807 CF 1,39 S1H6N3F1 2035 HF 0
S1H6N5F1 2441 CF 1,20 S1H6N6F1 2644 CF 0
S1H5N4 1930 C 1,17 S1H5N6F2 2482 CE 0
S1H5N4F1 2076 CF 1,14 S1H7N5F1Ac 2645 CF 0
S1H6N5F3 2733 CE 1,11 S1H5N5F3 2571 CE 0
S1H6N5 2295 C 1,06
S1H6N4F1 2238 CF 1,03
Proposed composition wherein the monosaccharide symbols are: S, NeuAc; G,
NeuGc, H, Hex; N,
HexNAc; F, dHex; Ac, acetyl ester.
2) Calculated mlz for [M-H]- ion rounded down to next integer.
3) N-glycan class symbols are: H, hybrid-type or monoantennary; C, complex-
type; 0, other type; F,
fucosylated; E, complex-fucosylated, wherein at least one fucose residue is
a1,2-, a1,3- or a1,4-linked.
4) `fold' is calculated as the relation of glycan signal intensities in hESC
compared to differentiated cell types
~hESC and St.3); -, not detected in differentiated cells; 0, not detected in
hESC.
) Association with differentiation type based on fold calculation: + low
association, ++ substantial
association, +++ high association.
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Table 3. N-glycan structural feature analysis based on proposed monosaccharide
compositions of
four hESC lines FES 21, FES 22, FES 29, and FES 30. The numbers refer to
percentage from either
neutral (A-E) or acidic (J-L) N-glycan pools, or from subfractions of
hybrid/monoantenary and
complex-type N-glycans (N>3, F-I and M-P). EB 29 and EB 30: embryoid bodies
derived from
hESC lines FES 29 and FES 30, respectively; st.3 29: stage 3 differentiated
cells derived from
hESC line FES 29. H: hexose; N: N-acetylhexosamine; F: deoxyhexose.
N 0) o
N N N M m M
t
~ LL LL LL w
A N=2 and 5:5H510 high-mannose type 84 73 79 79 73 72
B N=2 and 1<-H54 low-mannose type 5 11 7 8 12 12
C N=3 and H?2 hybrid/monoantennary 3 7 3 3 5 6
D N_4 and H_3 complex-type 6 9 10 10 8 8
R
~ E ther types 2 0 1 0 2 2
m
Z F F?1 ucosylation 8 11 10 10 14 15
M G F?2 complex fucosylation 1 0 2 2 2 2
z H N>H>-2 erminal N (N>H) 1 2 1 1 3 3
I N=H25 erminal N (N=H) 0 2 0 0 1 1
J N=3 and H?3 hybrid/monoantennary 8 2 5 9 13 14
K N_4 and H_3 complex-type 91 98 94 90 83 77
m L ther types 1 0 1 1 4 9
`- M F?1 ucosylation 85 96 75 78 83 86
`- N F?2 complex fucosylation 24 34 23 19 12 11
(n ~ M
nl
Z 0 N>H_3 erminal N (N>H) 10 8 6 5 10 10
P N=H?5 erminal N (N=H) 3 4 4 2 14 20
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Table 4. hESC, human embryonic stem cells; EB, embryoid bodies derived from
hESC; st.3, stage
3 differentiated cells derived from hESC; hEF, human fibroblast feeder cells;
mEF, murine
fibroblast feeder cells; BM MSC, bone-marrow derived mesenchymal stem cells;
OB, Osteoblast-
differentiated cells derived from BM MSC; CB MSC, cord blood derived
mesenchymal stem cells;
OB, adipocyte-differentiated cells derived from CB MSC; CB MNC, cord blood
mononuclear cells;
CD34+, CD133+, LIN-, and CD8-: subpopulations of CB MNC.
Hex5_gHexNAc2
U U U +
(including high-mannose type +
~ m M W W m M "' z
N-glycans) LU W N r s o m a m c o ~ ~
00 U U U U
Proposed composition m/z
Hex5HexNAc2 1257 + + + + + + + + + + + + + +
Hex6HexNAc2 1419 + + + + + + + + + + + + + +
Hex7HexNAc2 1581 + + + + + + + + + + + + + +
Hex8HexNAc2 1743 + + + + + + + + + + + + + +
Hex9HexNAc2 1905 + + + + + + + + + + + + + +
Hex,_4HexNAc2dHexo_,
U U U + +
(including low-mannose type cn m M LL W m U M z
N-glycans) t W r s ~ m a m o ~ c ~
00 U U U U
Proposed composition m/z
HexHexNAc2 609 + + + + + + + +
HexHexNAc2dHex 755 + + + + +
Hex2HexNAc2 771 + + + + + + + + + + + + + +
Hex2HexNAc2dHex 917 + + + + + + + + + + + + + +
Hex3HexNAc2 933 + + + + + + + + + + + + + +
Hex3HexNAc2dHex 1079 + + + + + + + + + + + + + +
Hex4HexNAc2 1095 + + + + + + + + + + + + + +
Hex4HexNAc2dHex 1241 + + + + + + + + + + + + + +
Hex10_12HexNAc2
(including glucosylated high- y m M W W ~ m ~ U + M
mannose type N-glycans) LU W ~ s ~ m a m m o ~ c ~
00 U U U U
Proposed composition mlz
HexlOHexNAc2 2067 + + + + + + + + + + + + + +
Hex11HexNAc2 2229 + + + + + + + + + + +
Hexl2HexNAc2 2391 + + + + + + + + + +
Hex5_9HexNAc2dHex,
(including fucosylated high- co m M W W m U M "' Z
mannose type N-glycans) t
m U U U U
Proposed composition mlz
Hex5HexNAc2dHex 1403 + + + + + + + + + + + + + +
Hex6HexNAc2dHex 1565 + + + + + + + + + +
Hex7HexNAc2dHex 1727 +
Hex,_gHexNAc,
U U U + +
(including soluble glycans) cn m M LL W m U Z M z_
L W N L ~ ~ O] a CO ~ J U
00 U U U U
Proposed composition mlz
Hex2HexNAc 568 + + + + + + +
Hex3HexNAc 730 + + + + + + + + +
Hex4HexNAc 892 + + + + + + + + + + + + + +
Hex5HexNAc 1054 + + + + + + + + + + + + + +
Hex6HexNAc 1216 + + + + + + + + + + + + + +
Hex7HexNAc 1378 + + + + + + + + + + + + + +
Hex8HexNAc 1540 + + + + + + + + + + + + +
Hex9HexNAc 1702 + + + + + + + + + +
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HexNAc=3 and Hex_2
(including hybrid-type and co m M_ W W ~ m ~ ~ ~ M Z
monoantennary N-glycans) t
Proposed composition mlz
Hex2HexNAc3 974 + + +
Hex2HexNAc3dHex 1120 + + + + + + + + +
Hex3HexNAc3 1136 + + + + + + + + + + + + + +
Hex2HexNAc3dHex2 1266 +
Hex3HexNAc3dHex 1282 + + + + + + + + + + + + + +
Hex4HexNAc3 1298 + + + + + + + + + + + + + +
Hex3HexNAc3dHex2 1428 + + + + + +
Hex4HexNAc3dHex 1444 + + + + + + + + + + + + + +
Hex5HexNAc3 1460 + + + + + + + + + + + + + +
Hex4HexNAc3dHex2 1590 + + + + + + + + +
Hex5HexNAc3dHex 1606 + + + + + + + + + + + + + +
Hex6HexNAc3 1622 + + + + + + + + + + + + + +
Hex5HexNAc3dHex2 1752 + + + +
Hex6HexNAc3dHex 1768 + + + + + + + + +
Hex7HexNAc3 1784 + + + + + + +
Hex8HexNAc3 1946 + +
HexNAc>_4 and Hex_3
(including complex-type N- y m M W W ~ m ~ ~ ~ M z
glycans) LU W ~ ~ s ~ m a m m o ~ c ~
m U U V U
Proposed composition mlz
Hex3HexNAc4 1339 + + + + + + + +
Hex3HexNAc4dHex 1485 + + + + + + + + + + + + + +
Hex4HexNAc4 1501 + + + + + + + + + +
Hex3HexNAc5 1542 + + + + + + + +
Hex4HexNAc4dHex 1647 + + + + + + + + + + + + + +
Hex5HexNAc4 1663 + + + + + + + + + + + + + +
Hex3HexNAc5dHex 1688 + + + + + + + + + + + + + +
Hex4HexNAx5 1704 + + + + + + + + + + + +
Hex4HexNAc4dHex2 1793 + + + + + + + +
Hex5HexNAc4dHex 1809 + + + + + + + + + + + + + +
Hex6HexNAc4 1825 + + + + + + + + + + +
Hex4HexNAc5dHex 1850 + + + + + + +
Hex5HexNAc5 1866 + + + + + + + + + + + +
Hex3HexNAc6dHex 1891 + + + + +
Hex5HexNAc4dHex2 1955 + + + + + + + + + + +
Hex6HexNAc4dHex 1971 + + + + + + + +
Hex7HexNAc4 1987 + + + + + + +
Hex4HexNAc5dHex2 1996 + + + + + + +
Hex5HexNAc5dHex 2012 + + + + + + + +
Hex6HexNAc5 2028 + + + + + + + + + + +
Hex5HexNAc4dHex3 2101 + + + + + + + + + + +
Hex6HexNAc4dHex2 2117 + +
Hex7HexNAc4dHex 2133 + + + +
Hex4HexNAc5dHex3 2142 + + + + + + +
HexBHexNAc4 2149 + + + + +
Hex5HexNAc5dHex2 2158 + + + +
Hex6HexNAc5dHex 2174 + + + + + + + + + +
Hex7HexNAc5 2190 + +
Hex6HexNAc6 2231 + +
Hex7HexNAc4dHex2 2279 + +
Hex5HexNAc5dHex3 2304 + + +
Hex6HexNAc5dHex2 2320 + + + + + +
Hex7HexNAc5dHex 2336 + +
Hex8HexNAc5 2352 + +
Hex7HexNAc6 2393 + + + + + +
Hex7HexNAc4dHex3 2425 + +
Hex6HexNAc5dHex3 2466 + + +
Hex8HexNAc5dHex 2498 + +
Hex7HexNAc6dHex 2539 + + + + +
Hex6HexNAc5dHex4 2612 + +
Hex8HexNAc7 2758 + +
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HexNAc_3 and dHex_1
(including fucosylated N- co
glycans) t
Proposed composition mlz
Hex2HexNAc3dHex 1120 + + + + + + + + +
Hex2HexNAc3dHex2 1266 +
Hex3HexNAc3dHex 1282 + + + + + + + + + + + + + +
Hex3HexNAc3dHex2 1428 + + + + + +
Hex4HexNAc3dHex 1444 + + + + + + + + + + + + + +
Hex4HexNAc3dHex2 1590 + + + + + + + + +
Hex5HexNAc3dHex 1606 + + + + + + + + + + + + + +
Hex5HexNAc3dHex2 1752 + + + +
Hex6HexNAc3dHex 1768 + + + + + + + + +
Hex3HexNAc4dHex 1485 + + + + + + + + + + + + + +
Hex4HexNAc4dHex 1647 + + + + + + + + + + + + + +
Hex3HexNAc5dHex 1688 + + + + + + + + + + + + + +
Hex4HexNAc4dHex2 1793 + + + + + + + +
Hex5HexNAc4dHex 1809 + + + + + + + + + + + + + +
Hex4HexNAc5dHex 1850 + + + + + + +
Hex3HexNAc6dHex 1891 + + + + +
Hex5HexNAc4dHex2 1955 + + + + + + + + + + +
Hex6HexNAc4dHex 1971 + + + + + + + +
Hex4HexNAc5dHex2 1996 + + + + + + +
Hex5HexNAc5dHex 2012 + + + + + + + +
Hex5HexNAc4dHex3 2101 + + + + + + + + + + +
Hex6HexNAc4dHex2 2117 + +
Hex7HexNAc4dHex 2133 + + + +
Hex4HexNAc5dHex3 2142 + + + + + + +
Hex5HexNAc5dHex2 2158 + + + +
Hex6HexNAc5dHex 2174 + + + + + + + + + +
Hex7HexNAc4dHex2 2279 + +
Hex5HexNAc5dHex3 2304 + + +
Hex6HexNAc5dHex2 2320 + + + + + +
Hex7HexNAc5dHex 2336 + +
Hex7HexNAc4dHex3 2425 + +
Hex6HexNAc5dHex3 2466 + + +
Hex8HexNAc5dHex 2498 + +
Hex7HexNAc6dHex 2539 + + + + +
Hex6HexNAc5dHex4 2612 + +
HexNAc_3 and dHex_2
(including multifucosylated N- cn m M W W m ~ ~ M + z
glycans) t w N L ~ ~ m a m ~ ~ CclJ
m U U V U
Proposed composition mlz
Hex2HexNAc3dHex2 1266 +
Hex3HexNAc3dHex2 1428 + + + + + +
Hex4HexNAc3dHex2 1590 + + + + + + + + +
Hex5HexNAc3dHex2 1752 + + + +
Hex4HexNAc4dHex2 1793 + + + + + + + +
Hex5HexNAc4dHex2 1955 + + + + + + + + + + +
Hex4HexNAc5dHex2 1996 + + + + + + +
Hex5HexNAc4dHex3 2101 + + + + + + + + + + +
Hex6HexNAc4dHex2 2117 + +
Hex4HexNAc5dHex3 2142 + + + + + + +
Hex5HexNAc5dHex2 2158 + + + +
Hex7HexNAc4dHex2 2279 + +
Hex5HexNAc5dHex3 2304 + + +
Hex6HexNAc5dHex2 2320 + + + + + +
Hex7HexNAc4dHex3 2425 + +
Hex6HexNAcSdHex3 2466 + + +
Hex6HexNAc5dHex4 2612 + +
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HexNAc>Hex_2
(terminal HexNAc, N>H) co
t w N L E g ~ O] a m G J U
m U U V U
Proposed composition mlz
Hex2HexNAc3 974 + + +
Hex2HexNAc3dHex 1120 + + + + + + + + +
Hex2HexNAc3dHex2 1266 +
Hex3HexNAc4 1339 + + + + + + + +
Hex3HexNAc4dHex 1485 + + + + + + + + + + + + + +
Hex3HexNAc5 1542 + + + + + + + +
Hex3HexNAc5dHex 1688 + + + + + + + + + + + + + +
Hex4HexNAx5 1704 + + + + + + + + + + + +
Hex4HexNAc5dHex 1850 + + + + + + +
Hex3HexNAc6dHex 1891 + + + + +
Hex4HexNAc5dHex2 1996 + + + + + + +
Hex4HexNAc5dHex3 2142 + + + + + + +
HexNAc=Hex_5
U U U + +
(terminal HexNAc, N=H) U) m M LL W m `~ U Z M M z_
t w N L E g ~ O] a m G ~ U
00 U U V U
Proposed composition m/z
Hex5HexNAc5 1866 + + + + + + + + + + + +
Hex5HexNAc5dHex 2012 + + + + + + + +
Hex5HexNAc5dHex2 2158 + + + +
Hex6HexNAc6 2231 + +
Hex5HexNAc5dHex3 2304 + + +
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Table 5. hESC, human embryonic stem cells; EB, embryoid bodies derived from
hESC; st.3, stage
3 differentiated cells derived from hESC; hEF, human fibroblast feeder cells;
mEF, murine
fibroblast feeder cells; BM MSC, bone-marrow derived mesenchymal stem cells;
OB, Osteoblast-
differentiated cells derived from BM MSC; CB MSC, cord blood derived
mesenchymal stem cells;
OB, adipocyte-differentiated cells derived from CB MSC; CB MNC, cord blood
mononuclear cells;
CD34+, CD133+, LIN-, and CD8-: subpopulations of CB MNC.
HexNAc=3 and Hex_2
(including hybrid-type and
r 0;
monoantennary N-glycans) LU W N L e m o U a U ~ U ~ ~
Proposed composition m/z
Hex3HexNAc3dHexSP 1338 +
Hex4HexNAc3SP 1354 + +
NeuAcHex3HexNAc3 1403 + + + + + + + + + +
NeuGcHex3HexNAc3 1419 +
Hex4HexNAc3dHexSP 1500 + + + + + + + + + +
Hex5HexNAc3SP 1516 + + + +
NeuAcHex3HexNAc3dHex 1549 + + + + + + + + + + + +
NeuAcHex3HexNAc3SP2 1563 + +
NeuAcHex4HexNAc3 1565 + + + + + + + + + + + + +
NeuGcHex4HexNAc3 1581 + + + + +
Hex4HexNAc3dHex2SP 1646 + +
Hex5HexNAc3dHexSP 1662 +
Hex6HexNAc3SP and/or 1678 + + + + + + + + + + + + +
NeuAc2Hex2HexNAc3dHex
NeuAc2Hex3HexNAc3 1694 +
NeuAcHex3HexNAc3dHexSP2 1709 + +
NeuAcHex4HexNAc3dHex 1711 + + + + + + + + + + + + + +
NeuAcHex5HexNAc3 and/or 1727 + + + + + + + + + + + + +
NeuGcHex4HexNAc3dHex
NeuGcHex5HexNAc3 1743 +
NeuAcHex4HexNAc3dHexSP 1791 + + + + + +
Hex5HexNAc3dHex2SP 1808 +
NeuAc2Hex3HexNAc3dHex 1840 + + + + + + +
NeuAc2Hex4HexNAc3 1856 + +
NeuAcHex4HexNAc3dHex2 1857 + +
NeuAcHex5HexNAc3dHex and/or 1873 + + + + + + + + + + + + + +
NeuGcHex4HexNAc3dHex2
NeuAcHex6HexNAc3 1889 + + + + + + + + + + + + +
Hex8HexNAc3SP and/or 2002 + + + + + + + + + +
NeuAc2Hex4HexNAc3dHex
NeuAcHex4HexNAc3dHex3 2003 + +
NeuAc2Hex5HexNAc3 and/or 2018 + + + + + + +
NeuGcNeuAcHex4HexNAc3dHex
NeuAcHex5HexNAc3dHex2 2019 + + +
NeuGcNeuAcHex5HexNAc3 and/or 2034 +
NeuGc2Hex4HexNAc3dHex
NeuAcHex6HexNAc3dHex 2035 + + + + + + + + + +
NeuGc2Hex5HexNAc3 2050 +
NeuAcHex7HexNAc3 2051 + + + + + +
NeuAc2Hex4HexNAc3dHexSP and/or 2082 + + +
Hex8HexNAc3SP2
NeuAcHex6HexNAc3dHexSP 2115 +
Hex8HexNAc3dHexSP and/or 2148 +
NeuAc2 Hex4H exNAc3d Hex2
NeuAcHex8HexNAc3SP and/or 2293 +
NeuAc3Hex4HexNAc3dHex
NeuAc2Hex5HexNAc3dHex2 and/or 2310 +
NeuGcNeuAcHex4HexNAc3dHex3
NeuAc3Hex5HexNAc3SP 2389 +
NeuAc2Hex5HexNAc3dHex2SP 2390 + + + + + + + + + +
NeuAc2Hex6HexNAc3dHexSP 2406 + + +
NeuAcHex8HexNAc3dHexSP and/or 2439 +
NeuAc3 Hex4H exNAc3d Hex2
NeuAcHex9HexNAc3dHex 2521 +
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HexNAc>_4 and Hex_3
(including complex-type N- co m M W W ~ m ~ ~ ~ M Z
glycans) t W ~ ~ ~ ~ m a m m o ~ c ~
m U U V U
Proposed composition mlz
Hex4HexNAc4SP 1557 + + + +
NeuAcHex3HexNAc4 1606 +
Hex4HexNAc4SP2 1637 + + + + + + + +
Hex4HexNAc4dHexSP 1703 + + +
Hex4HexNAc4SP3 and/or 1717 +
Hex7HexNAc2SP2
Hex5HexNAc4SP 1719 + + + + + +
NeuAcHex3HexNAc4dHex 1752 +
NeuAcHex4HexNAc4 1768 + + + + + + + + + + + +
NeuGcHex4HexNac4 1784 + +
Hex5HexNAc4SP2 and/or 1799 + + +
Hex8HexNAc2SP
NeuAcHex3HexNac5 1809 +
NeuGcHex3HexNAc5 1825 + +
Hex5HexNAc4dHexSP 1865 + + + + + + + + + + +
Hex6HexNAc4SP 1881 +
Hex4HexNAc5dHexSP 1906 + +
NeuAcHex4HexNAc4dHex 1914 + + + + + + + + + + + + +
NeuAcHex4HexNAc4SP2 1928 + +
NeuAcHex5HexNAc4 1930 + + + + + + + + + + + + + +
NeuGcHex5HexNAc4 1946 + + + + + + + +
NeuAcHex4HexNAc5 1971 + + + + + + +
NeuAcHex5HexNAc4Ac 1972 +
Hex5HexNAc5SP2 2002 + + + + + + +
NeuAcHex5HexNAc4SP 2010 + +
Hex5HexNAc4dHex2SP 2011 +
NeuGcHex5HexNAc4SP 2026 +
Hex6HexNAc4dHexSP 2027 + +
Hex7HexNAc4SP and/or
Hex4HexNAc6SP2 and/or 2043 +
NeuAc2Hex3HexNAc4dHex
NeuAcHex4HexNAc5SP 2051 + + + + +
Hex4HexNAc5dHex2SP 2052 + + + +
NeuAc2Hex4HexNAc4 2059 + +
NeuAcHex4HexNAc4dHex2 2060 + + + + + +
NeuAcHex4HexNAc4dHexSP2 2074 + +
NeuAcHex5HexNAc4dHex 2076 + + + + + + + + + + + + + +
NeuAcHex6HexNAc4 and/or 2092 + + + + + + + + + + + +
NeuGcHex5HexNAc4dHex
NeuAcHex3HexNAc5dHex2 and/or 2101 +
NeuAc2Hex4HexNAc4Ac
NeuGcHex6HexNAc4 2108 +
NeuAcHex4HexNAc5dHex 2117 + + + + + + + + +
Hex4HexNAc5dHex2SP2 2132 +
NeuAcHex5HexNAc5 2133 + + + + + + + + + +
NeuAc2Hex4HexNAc4SP 2139
NeuAcHex5HexNAc4dHexSP 2156 + + + + + + +
Hex5HexNAc4dHex3SP 2157 +
Hex6HexNAc5SP2 2164 + + +
Hex6HexNAc4dHex2SP and/or 2173 +
Hex3HexNAc6dHex2SP2
NeuAcHex4HexNAc6 2174 + + + + + +
NeuAc3Hex3HexNAc4 and/or
NeuGcHex6HexNAc4SP and/or 2188 + +
NeuAc2NeuGcHex2HexNAc4dHex
NeuAc2Hex3HexNAc4dHex2 and/or
Hex7HexNAc4dHexSP and/or 2189 + +
Hex4HexNAc6dHexSP2
NeuAc2Hex4HexNAc4dHex 2205 +
NeuAc2Hex4HexNAc4SP2 2219 +
NeuAc2Hex5HexNAc4 2221 + + + + + + + + + + + + + +
NeuAcHex5HexNAc4dHex2 2222 + + + + + + + + + + + + + +
Hex6HexNAc5dHexSP 2230 + + + +
NeuGcNeuAcHex5HexNAcA 2237 + + + + + + +
NeuAcHex6HexNAc4dHex and/or 2238 + + + + + + + + + + + + +
NeuGcHex5HexNAc4dHex2
NeuAc2Hex3HexNAc5dHex and/or 2246 + + + +
Hex7HexNAc5SP
NeuGc2Hex5HexNAc4 2253 + + + + + +
NeuAcHex7HexNAc4 andlor 2254 + + + + + + + + + +
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NeuGcHex6HexNAc4dHex
NeuAc2Hex4HexNAc5 2262 +
NeuAcHex4HexNAc5dHex2 and/or 2263 + + +
NeuAc2Hex5HexNAc4Ac
NeuAcHex5HexNAc5dHex 2279 + + + + + + + + + + + + + +
NeuAc2Hex4HexNAc4dHexSP and/or 2285 +
Hex11HexNAc2SP
NeuAcHex6HexNAc5 2295 + + + + + + + + + + + + +
NeuAc2Hex5HexNAc4SP 2301 +
NeuAcHex5HexNAc4dHex2SP 2302 +
NeuAc2Hex5HexNAc4Ac2 2305 +
Hex6HexNAc4dHex3SP and/or 2319 + + +
NeuGcNeuAcHex3HexNAc6
NeuAcHex4HexNAc6dHex 2320 + +
NeuAcHex5HexNAc5dHexAc 2321 + +
Hex7HexNAc4dHex2SP andlor 2335 + +
Hex4HexNAc6dHex2SP2
NeuAcHex5HexNAc6 2336 + +
NeuAc3Hex4HexNac4 2350 +
NeuAc2Hex4HexNAc4dHexSP 2365 + + +
NeuAc2Hex5HexNAc4dHex 2367 + + + + + + + + + + + + + +
NeuAcHex5HexNAc4dHex3 2368 + + + + + + + + + + + + +
NeuAc2Hex6HexNAc4 and/or 2383 + + + + + + + + +
NeuGcNeuAcHex5HexNAcAdHex
NeuAcHex6HexNAc4dHex2 and/or 2384 + + + + + + +
NeuGcHex5HexNAc4dHex3
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + +
Hex7HexNAc5dHexSP
NeuAcHex3HexNAc5dHex4 2393 +
NeuGc2Hex5HexNAc4dHex 2399 + + +
NeuAcHex4HexNAc6dHexSP and/or
NeuGcHex6HexNAc4dHex2 and/or 2400 +
NeuAcHex7HexNAc4dHex
NeuAc2Hex4HexNAc5dHex 2408 + + +
NeuAcHex4HexNAc5dHex3 and/or 2409 + +
NeuAc2Hex5HexNAc4dHexAc
NeuAc2Hex5HexNAc5 2424 + + + + +
NeuAcHex5HexNAc5dHex2 2425 + + + + + + + + + +
NeuAcHex6HexNAc5dHex 2441 + + + + + + + + + + + + + +
NeuAc2Hex5HexNAc4dHexSP 2447 + + + + + + +
NeuAcHex5HexNAc4dHex3SP 2448 + + + + +
NeuAcHex7HexNAc5 and/or 2457 + + + + +
NeuGcHex6HexNAc5dHex
NeuGcHex7HexNAc5 2473 + +
NeuAcHex5HexNAc6dHex 2482 +
NeuAcHex4HexNAc5dHex3SP 2489 + +
Hex6HexNAc7SP 2490 +
NeuAc3Hex5HexNAc4 2512 + + + +
NeuAc2Hex5HexNAc4dHex2 2513 + + + + + + +
NeuAcHex5HexNAc4dHex4 2514 + +
NeuAcHex6HexNAc5dHexSP and/or 2521 + + + +
NeuAc3 Hex2H exNAc5d Hex2
Hex6HexNAc5dHex3SP 2522 + +
NeuGcNeuAc2Hex5HexNAc4 2528 + + + + +
NeuAc2Hex6HexNAc4dHex and/or 2529 + + + +
NeuGcNeuAcHex5HexNAcAdHex2
NeuGc2NeuAcHex5HexNAc4 2544 + + + + + +
NeuGc2Hex5HexNAcAdHex2 and/or 2545 + + +
NeuGcNeuAcHex6HexNAcAdHex
NeuGc3Hex5HexNAc4 2560 + + + +
NeuGc2Hex6HexNAcAdHex 2561 +
NeuAc2Hex5HexNAc5dHex 2570 + + + + + + + +
NeuAcHex5HexNAc5dHex3 2571 + + + + + + + +
NeuAc2Hex6HexNAc5 2586 + + + + + + + + + + +
NeuAcHex6HexNAc5dHex2 2587 + + + + + + + + + + + +
Hex7HexNAc6dHexSP 2595 +
NeuGcNeuAcHex6HexNAc5 2602 + + +
NeuAcHex7HexNAc5dHex and/or 2603 + + + + + + +
NeuGcHex6HexNAc5dHex2
NeuAcHex8HexNAc5 and/or 2619 + + +
NeuGcHex7HexNAc5dHex
NeuAc2Hex5HexNAc6 2627 +
NeuGcHex8HexNAc5 and/or 2635 + +
NeuAcHex4HexNAc5dHex4SP
NeuAcHex6HexNAc6dHex 2644 + + + + + + + + + + +
NeuAc2Hex5HexNAc4dHex3 2659 + +
NeuAcHex7HexNAc6 2660 + + + + + + + + +
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
159
NeuGcNeuAc2Hex5HexNAc4dHex 2674 + +
and/or NeuAc3Hex6HexNAc4
NeuAc2Hex4HexNAc5dHex2SP2 2714 + + + +
NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAc3Hex5HexNAc5
NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAc2Hex6HexNAc5dHex 2732 + + + + + + + + + + + + +
NeuAcHex6HexNAc5dHex3 2733 + + + + + + + + + + + + +
NeuGcNeuAcHex6HexNAc5dHex 2748 +
NeuAcHex8HexNAc5dHex 2765 +
NeuGcHex8HexNAc5dHex and/or 2781 +
NeuAcHex9HexNAc5
NeuAcHex6HexNAc6dHex2 2791 + + + +
Hex6HexNAc6dHex3SP2 2805 +
NeuAcHex7HexNAc6dHex 2807 + + + + + + + + + + + + +
NeuAc2Hex6HexNAc5dHexSP 2812 + + + + +
NeuAcHex6HexNAc5dHex3SP 2813 +
NeuGcNeuAc3Hex5HexNAc4 2819 +
NeuAc3Hex6HexNAc4dHex and/or 2820 +
NeuGcNeuAc2Hex5HexNAc4dHex2
NeuAc3Hex6HexNAc5 2878 + + + + + + + + + + + +
NeuAc2Hex6HexNAc5dHex2 2879 + + + + + + + + + + + + +
NeuAcHex6HexNAc5dHex4 2880 + + + + +
NeuGcNeuAc2Hex6HexNAc5 2894 + +
NeuAc2Hex7HexNAc5dHex and/or 2895 + +
NeuGcNeuAcHex6HexNAc5dHex2
NeuAc3Hex6HexNAc4dHexSP and/or 2900 +
NeuGcNeuAc2Hex5HexNAc4dHex2SP
NeuGc2Hex6HexNAc5dHex2 2911 +
NeuAc2Hex5HexNAc6dHex2 2920 +
NeuGc3Hex6HexNAc5 2925 +
NeuGcNeuAc2Hex5HexNAc6 2935 +
NeuAc2Hex6HexNAc6dHex and/or 2936 + + + + + + +
NeuGcNeuAcHex5HexNAc6dHex2
NeuAcHex6HexNAc6dHex3 2937 + +
NeuGc2NeuAcHex5HexNAc6 and/or 2951 +
NeuAc3 Hex5H exNAc4d Hex3
NeuAc2Hex7HexNAc6 2952 + + + + + +
NeuAcHex7HexNAcBdHex2 2953 + + + + + + + +
Hex8HexNAc7dHexSP 2961 +
NeuAc2Hex4HexNAc7dHex2 2961 +
NeuAcHex7HexNAc7dHex 3010 + + +
NeuAc3Hex6HexNAc5dHex 3024 + + + + + + + + + + + +
NeuAc2Hex6HexNAc5dHex3 3025 + + + + + + + + + + +
NeuAcHex8HexNAc7 3026 + + + + + +
NeuGc3Hex6HexNAc5dHex and/or 3072 +
NeuGc2NeuAcHex7HexNAc5
NeuAc2Hex6HexNAc6dHex2 3082 +
NeuAc2Hex7HexNAc6dHex 3098 + + + + + + + + + + + + +
NeuAcHex7HexNAc6dHex3 3099 + + + + + + + + + + + +
NeuAc3Hex6HexNAc5dHexSP 3104 + +
NeuAc2Hex6HexNAc5dHex3SP 3105 + +
NeuAc3Hex6HexNAc5dHex2 3170 + +
NeuAc2Hex6HexNAc5dHex4 3171 + + + + + +
NeuAcHex8HexNAc7dHex 3172 + + + + + + + + + + +
NeuAc3Hex6HexNAc6dHex 3227 + +
NeuAc2Hex6HexNAc6dHex3 3228 +
NeuAc3Hex7HexNAc6 3243 + + +
NeuAc2Hex7HexNAc6dHex2 3244 + + + + +
NeuAcHex7HexNAc6dHex4 3245 + + + + + +
NeuAc2Hex7HexNAc7dHex 3301 +
NeuAcHex7HexNAc7dHex3 3302 +
NeuAc2Hex8HexNAc7 3317 + + + +
NeuAcHex8HexNAc7dHex2 3318 + + +
NeuAc3Hex7HexNAc6dHex 3389 + + + + + + +
NeuAc2Hex7HexNAc6dHex3 3390 + + + + + + + + + +
NeuAcHex7HexNAc6dHex5 and/or 3391 + + +
NeuAcHex9HexNAc8
NeuAc2Hex8HexNAc7dHex 3463 + + + + + + + + +
NeuAcHex8HexNAc7dHex3 3464 + + + + + +
NeuAc2Hex7HexNAc6dHex4 3536 + + + + + +
NeuAcHex9HexNAc8dHex 3537 + + + + +
NeuAc3Hex8HexNAc7 3608 + +
NeuAc2Hex8HexNac7dHex2 3609 + + +
NeuAcHex8HexNac7dHex4 3610 + + + +
NeuAc4Hex7HexNAc6dHex 3680 + + +
NeuAc3Hex7HexNAc6dHex3 3681 + + + + + + +
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
160
NeuAc2Hex9HexNAc8 3682 + + +
NeuAcHex9HexNAc8dHex2 3683 + + +
NeuAc3Hex8HexNAc7dHex 3754 + + + +
NeuAc2Hex8HexNAc7dHex3 3755 + + + + + +
NeuAcHex10HexNAc9 and/or 3756 + + + +
NeuAcHex8HexNAc7dHex5
NeuAc4Hex6HexNAc8 3778 +
NeuAc3Hex7HexNAc6dHex4 3827 + +
NeuAc2Hex9HexNAc8dHex 3828 + + + +
NeuAcHex9HexNAc8dHex3 3829 + + + +
NeuAc2Hex8HexNAc7dHex4 3901 + + +
NeuAc2Hex9HexNAc8dHex2 3974 + +
NeuAcHex9HexNAc8dHex4 3975 + +
NeuAc4Hex8HexNAc7dHex 4045 +
NeuAc3Hex8HexNAc7dHex3 4046 + +
NeuAc2HexlOHexNAc9 and/or 4047 + +
NeuAc2 Hex8H exNAc7d Hex5
NeuAc3Hex9HexNAc8dHex 4119 +
NeuAc2Hex9HexNAc8dHex3 4120 +
HexNAc_3 and dHex_1
U U U + +
(including fucosylated N- cn m M W w ~ m ~ ~ ~ M "' z
glycans) t W ~ r s ~ m a m ~ o ~ c ~
m U U U U
Proposed composition mlz
Hex3HexNAc3dHexSP 1338 +
Hex4HexNAc3dHexSP 1500 + + + + + + + + + +
NeuAcHex3HexNAc3dHex 1549 + + + + + + + + + + + +
Hex4HexNAc3dHex2SP 1646 + +
Hex5HexNAc3dHexSP 1662 +
Hex6HexNAc3SP and/or 1678 + + + + + + + + + + + + +
NeuAc2Hex2HexNAc3dHex
NeuAcHex3HexNAc3dHexSP2 1709 + +
NeuAcHex4HexNAc3dHex 1711 + + + + + + + + + + + + + +
NeuAcHex5HexNAc3 and/or 1727 + + + + + + + + + + + + +
NeuGcHex4HexNAc3dHex
NeuAcHex4HexNAc3dHexSP 1791 + + + + + +
Hex5HexNAc3dHex2SP 1808 +
NeuAc2Hex3HexNAc3dHex 1840 + + + + + + +
NeuAcHex4HexNAc3dHex2 1857 + +
NeuAcHex5HexNAc3dHex and/or 1873 + + + + + + + + + + + + + +
NeuGcHex4HexNAc3dHex2
Hex8HexNAc3SP and/or 2002 + + + + + + + + + +
NeuAc2Hex4HexNAc3dHex
NeuAcHex4HexNAc3dHex3 2003 + +
NeuAc2Hex5HexNAc3 and/or 2018 + + + + + + +
NeuGcNeuAcHex4HexNAc3dHex
NeuAcHex5HexNAc3dHex2 2019 + + +
NeuGcNeuAcHex5HexNAc3 and/or 2034 +
NeuGc2Hex4HexNAc3dHex
NeuAcHex6HexNAc3dHex 2035 + + + + + + + + + +
NeuAc2Hex4HexNAc3dHexSP and/or 2082 + + +
Hex8HexNAc3SP2
NeuAcHex6HexNAc3dHexSP 2115 +
Hex8HexNAc3dHexSP and/or 2148 +
NeuAc2 Hex4H exNAc3d Hex2
NeuAcHex8HexNAc3SP and/or 2293 +
NeuAc3Hex4HexNAc3dHex
NeuAc2Hex5HexNAc3dHex2 and/or 2310 +
NeuGcNeuAcHex4HexNAc3dHex3
NeuAc2Hex5HexNAc3dHex2SP 2390 + + + + + + + + + +
NeuAc2Hex6HexNAc3dHexSP 2406 + + +
NeuAcHex8HexNAc3dHexSP and/or 2439 +
NeuAc3 Hex4H exNAc3d Hex2
NeuAcHex9HexNAc3dHex 2521 +
Hex4HexNAc4dHexSP 1703 + + +
NeuAcHex3HexNAc4dHex 1752 +
Hex5HexNAc4dHexSP 1865 + + + + + + + + + + +
Hex4HexNAc5dHexSP 1906 + +
NeuAcHex4HexNAc4dHex 1914 + + + + + + + + + + + + +
Hex5HexNAc4dHex2SP 2011 +
Hex6HexNAc4dHexSP 2027 + +
Hex7HexNAc4SP and/or
Hex4HexNAc6SP2 and/or 2043 +
NeuAc2Hex3HexNAc4dHex
Hex4HexNAc5dHex2SP 2052 + + + +
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
161
NeuAcHex4HexNAc4dHex2 2060 + + + + + +
NeuAcHex4HexNAc4dHexSP2 2074 + +
NeuAcHex5HexNAc4dHex 2076 + + + + + + + + + + + + + +
NeuAcHex6HexNAc4 and/or 2092 + + + + + + + + + + + +
NeuGcHex5HexNAc4dHex
NeuAcHex3HexNAc5dHex2 and/or 2101 +
NeuAc2Hex4HexNAc4Ac
NeuAcHex4HexNAc5dHex 2117 + + + + + + + + +
Hex4HexNAc5dHex2SP2 2132 +
NeuAcHex5HexNAc4dHexSP 2156 + + + + + + +
Hex5HexNAc4dHex3SP 2157 +
Hex6HexNAc4dHex2SP and/or 2173 +
Hex3HexNAc6dHex2SP2
NeuAc3Hex3HexNAc4 and/or
NeuGcHex6HexNAc4SP and/or 2188 + +
NeuAc2NeuGcHex2HexNAc4dHex
NeuAc2Hex3HexNAc4dHex2 and/or
Hex7HexNAc4dHexSP and/or 2189 + +
Hex4HexNAc6dHexSP2
NeuAc2Hex4HexNAc4dHex 2205 +
NeuAcHex5HexNAc4dHex2 2222 + + + + + + + + + + + + + +
Hex6HexNAc5dHexSP 2230 + + + +
NeuAcHex6HexNAc4dHex and/or 2238 + + + + + + + + + + + + +
NeuGcHex5HexNAc4dHex2
NeuAc2Hex3HexNAc5dHex and/or 2246 + + + +
Hex7HexNAc5SP
NeuAcHex7HexNAc4 andlor 2254 + + + + + + + + + +
NeuGcHex6HexNAc4dHex
NeuAcHex4HexNAc5dHex2 and/or 2263 + + +
NeuAc2Hex5HexNAc4Ac
NeuAcHex5HexNAc5dHex 2279 + + + + + + + + + + + + + +
NeuAc2Hex4HexNAc4dHexSP and/or 2285 +
Hex11HexNAc2SP
NeuAcHex5HexNAc4dHex2SP 2302 +
Hex6HexNAc4dHex3SP and/or 2319 + + +
NeuGcNeuAcHex3HexNAc6
NeuAcHex4HexNAc6dHex 2320 + +
NeuAcHex5HexNAc5dHexAc 2321 + +
Hex7HexNAc4dHex2SP and/or 2335 + +
Hex4HexNAc6dHex2SP2
NeuAc2Hex4HexNAc4dHexSP 2365 + + +
NeuAc2Hex5HexNAc4dHex 2367 + + + + + + + + + + + + + +
NeuAcHex5HexNAc4dHex3 2368 + + + + + + + + + + + + +
NeuAc2Hex6HexNAc4 and/or 2383 + + + + + + + + +
NeuGcNeuAcHex5HexNAcAdHex
NeuAcHex6HexNAc4dHex2 and/or 2384 + + + + + + +
NeuGcHex5HexNAc4dHex3
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + +
Hex7HexNAc5dHexSP
NeuAcHex3HexNAc5dHex4 2393 +
NeuGc2Hex5HexNAc4dHex 2399 + + +
NeuAcHex4HexNAc6dHexSP and/or
NeuGcHex6HexNAc4dHex2 and/or 2400 +
NeuAcHex7HexNAc4dHex
NeuAc2Hex4HexNAc5dHex 2408 + + +
NeuAcHex4HexNAc5dHex3 and/or 2409 + +
NeuAc2Hex5HexNAc4dHexAc
NeuAcHex5HexNAc5dHex2 2425 + + + + + + + + + +
NeuAcHex6HexNAc5dHex 2441 + + + + + + + + + + + + + +
NeuAc2Hex5HexNAc4dHexSP 2447 + + + + + + +
NeuAcHex5HexNAc4dHex3SP 2448 + + + + +
NeuAcHex7HexNAc5 and/or 2457 + + + + +
NeuGcHex6HexNAc5dHex
NeuAcHex5HexNAc6dHex 2482 +
NeuAcHex4HexNAc5dHex3SP 2489 + +
NeuAc2Hex5HexNAc4dHex2 2513 + + + + + + +
NeuAcHex5HexNAc4dHex4 2514 + +
NeuAcHex6HexNAc5dHexSP and/or 2521 + + + +
NeuAc3 Hex2H exNAc5d Hex2
Hex6HexNAc5dHex3SP 2522 + +
NeuAc2Hex6HexNAc4dHex and/or 2529 + + + +
NeuGcNeuAcHex5HexNAcAdHex2
NeuGc2Hex5HexNAcAdHex2 and/or 2545 + + +
NeuGcNeuAcHex6HexNAcAdHex
NeuGc2Hex6HexNAc4dHex 2561 +
NeuAc2Hex5HexNAc5dHex 2570 + + + + + + + +
NeuAcHex5HexNAc5dHex3 2571 + + + + + + + +
NeuAcHex6HexNAc5dHex2 2587 + + + + + + + + + + + +
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
162
Hex7HexNAc6dHexSP 2595 +
NeuAcHex7HexNAc5dHex and/or 2603 + + + + + + +
NeuGcHex6HexNAc5dHex2
NeuAcHex8HexNAc5 andlor 2619 + + +
NeuGcHex7HexNAc5dHex
NeuGcHex8HexNAc5 and/or 2635 + +
NeuAcHex4HexNAc5dHex4SP
NeuAcHex6HexNAc6dHex 2644 + + + + + + + + + +
NeuAc2Hex5HexNAc4dHex3 2659 + +
NeuGcNeuAc2Hex5HexNAc4dHex 2674 + +
and/or NeuAc3Hex6HexNAc4
NeuAc2Hex4HexNAc5dHex2SP2 2714 + + + +
NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAc3Hex5HexNAc5
NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAc2Hex6HexNAc5dHex 2732 + + + + + + + + + + + + +
NeuAcHex6HexNAc5dHex3 2733 + + + + + + + + + + + + +
NeuGcNeuAcHex6HexNAc5dHex 2748 +
NeuAcHex8HexNAc5dHex 2765 +
NeuAcHex6HexNAc6dHex2 2791 + + + +
Hex6HexNAc6dHex3SP2 2805 +
NeuAcHex7HexNAc6dHex 2807 + + + + + + + + + + + + +
NeuAc2Hex6HexNAc5dHexSP 2812 + + + + +
NeuAcHex6HexNAc5dHex3SP 2813 +
NeuAc3Hex6HexNAc4dHex and/or 2820 +
Neu GcNeuAc2Hex5HexNAc4d Hex2
NeuAc2Hex6HexNAc5dHex2 2879 + + + + + + + + + + + + +
NeuAcHex6HexNAc5dHex4 2880 + + + + +
NeuAc2Hex7HexNAc5dHex and/or 2895 + +
NeuGcNeuAcHex6HexNAc5dHex2
NeuAc3Hex6HexNAc4dHexSP and/or 2900 +
NeuGcNeuAc2Hex5HexNAc4dHex2SP
NeuGc2Hex6HexNAc5dHex2 2911 +
NeuAc2Hex5HexNAc6dHex2 2920 +
NeuGcNeuAc2Hex5HexNAc6 2935 +
NeuAc2Hex6HexNAc6dHex and/or 2936 + + + + + + +
NeuGcNeuAcHex5HexNAc6dHex2
NeuAcHex6HexNAcBdHex3 2937 + +
NeuGc2NeuAcHex5HexNAc6 and/or 2951 +
NeuAc3 Hex5H exNAc4d Hex3
NeuAcHex7HexNAc6dHex2 2953 + + + + + + + +
Hex8HexNAc7dHexSP 2961 +
NeuAc2Hex4HexNAc7dHex2 2961 +
NeuAcHex7HexNAc7dHex 3010 + + +
NeuAc3Hex6HexNAc5dHex 3024 + + + + + + + + + + + +
NeuAc2Hex6HexNAc5dHex3 3025 + + + + + + + + + + +
NeuGc3Hex6HexNAc5dHex and/or 3072 +
NeuGc2NeuAcHex7HexNAc5
NeuAc2Hex6HexNAc6dHex2 3082 +
NeuAc2Hex7HexNAc6dHex 3098 + + + + + + + + + + + + +
NeuAcHex7HexNAc6dHex3 3099 + + + + + + + + + + + +
NeuAc3Hex6HexNAc5dHexSP 3104 + +
NeuAc2Hex6HexNAc5dHex3SP 3105 + +
NeuAc3Hex6HexNAc5dHex2 3170 + +
NeuAc2Hex6HexNAc5dHex4 3171 + + + + + +
NeuAcHex8HexNAc7dHex 3172 + + + + + + + + + + +
NeuAc3Hex6HexNAc6dHex 3227 + +
NeuAc2Hex6HexNAc6dHex3 3228 +
NeuAc2Hex7HexNAc6dHex2 3244 + + + + +
NeuAcHex7HexNAc6dHex4 3245 + + + + + +
NeuAc2Hex7HexNAc7dHex 3301 +
NeuAcHex7HexNAc7dHex3 3302 +
NeuAcHex8HexNAc7dHex2 3318 + + +
NeuAc3Hex7HexNAc6dHex 3389 + + + + + + +
NeuAc2Hex7HexNAc6dHex3 3390 + + + + + + + + + +
NeuAcHex7HexNAc6dHex5 and/or 3391 + + +
NeuAcHex9HexNAcB
NeuAc2Hex8HexNAc7dHex 3463 + + + + + + + + +
NeuAcHex8HexNAc7dHex3 3464 + + + + + +
NeuAc2Hex7HexNAc6dHex4 3536 + + + + + +
NeuAcHex9HexNAc8dHex 3537 + + + + +
NeuAc2Hex8HexNac7dHex2 3609 + i+H +
NeuAcHex8HexNac7dHex4 3610 + + +
NeuAc4Hex7HexNAc6dHex 3680 + +
NeuAc3Hex7HexNAc6dHex3 3681 + + + + +
NeuAcHex9HexNAc8dHex2 3683 + + +
NeuAc3Hex8HexNAc7dHex 3754 + + +
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
163
NeuAc2Hex8HexNAc7dHex3 3755 + + + + + +
NeuAcHexlOHexNAc9 and/or 3756 + + + +
NeuAcHex8HexNAc7dHex5
NeuAc3Hex7HexNAc6dHex4 3827 + +
NeuAc2Hex9HexNAc8dHex 3828 + + + +
NeuAcHex9HexNAc8dHex3 3829 + + + +
NeuAc2Hex8HexNAc7dHex4 3901 + + +
NeuAc2Hex9HexNAc8dHex2 3974 + +
NeuAcHex9HexNAc8dHex4 3975 + +
NeuAc4Hex8HexNAc7dHex 4045 +
NeuAc3Hex8HexNAc7dHex3 4046 + +
NeuAc2HexlOHexNAc9 and/or 4047 + +
NeuAc2 Hex8H exNAc7d Hex5
NeuAc3Hex9HexNAc8dHex 4119 +
NeuAc2Hex9HexNAc8dHex3 4120 +
HexNAc>_3 and dHex>_1
(including multifucosylated N- y m M W W ~ m ~ ~ ~ M M z_
glycans) L W N L ~ m o ~ a ~ ~ ~ ~ ~1
Proposed composition m/z
Hex5HexNAc3dHex2SP 1808 +
NeuAcHex4HexNAc3dHex2 1857 + +
NeuAcHex5HexNAc3dHex and/or 1873 + + + + + + + + + + + + + +
NeuGcHex4HexNAc3dHex2
NeuAcHex4HexNAc3dHex3 2003 + +
NeuAcHex5HexNAc3dHex2 2019 + + +
Hex8HexNAc3dHexSP and/or 2148 +
NeuAc2 Hex4H exNAc3d Hex2
NeuAc2Hex5HexNAc3dHex2 and/or 2310 +
NeuGcNeuAcHex4HexNAc3dHex3
NeuAc2Hex5HexNAc3dHex2SP 2390 + + + + + + + + + +
NeuAcHex8HexNAc3dHexSP and/or 2439 +
NeuAc3 Hex4H exNAc3d Hex2
Hex5HexNAc4dHex2SP 2011 +
Hex4HexNAc5dHex2SP 2052 + + + +
NeuAcHex4HexNAc4dHex2 2060 + + + + + +
NeuAcHex3HexNAc5dHex2 and/or 2101 +
NeuAc2Hex4HexNAc4Ac
Hex4HexNAc5dHex2SP2 2132 +
Hex5HexNAc4dHex3SP 2157 +
Hex6HexNAc4dHex2SP and/or 2173 +
Hex3HexNAc6dHex2SP2
NeuAcHex5HexNAc4dHex2 2222 + + + + + + + + + + + + + +
NeuAcHex6HexNAc4dHex and/or 2238 + + + + + + + + + + + + +
NeuGcHex5HexNAc4dHex2
NeuAcHex4HexNAc5dHex2 and/or 2263 + + +
NeuAc2Hex5HexNAc4Ac
NeuAcHex5HexNAc4dHex2SP 2302 +
Hex6HexNAc4dHex3SP and/or 2319 + + +
NeuGcNeuAcHex3HexNAc6
Hex7HexNAc4dHex2SP and/or 2335 + +
Hex4HexNAc6dHex2SP2
NeuAcHex5HexNAc4dHex3 2368 + + + + + + + + + + + + +
NeuAcHex6HexNAc4dHex2 and/or 2384 + + + + + + +
NeuGcHex5HexNAc4dHex3
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + +
Hex7HexNAc5dHexSP
NeuAcHex3HexNAc5dHex4 2393 +
NeuAcHex4HexNAc6dHexSP and/or
NeuGcHex6HexNAc4dHex2 and/or 2400 +
NeuAcHex7HexNAc4dHex
NeuAcHex4HexNAc5dHex3 and/or 2409 + +
NeuAc2Hex5HexNAc4dHexAc
NeuAcHex5HexNAc5dHex2 2425 + + + + + + + + + +
NeuAcHex5HexNAc4dHex3SP 2448 + + + + +
NeuAcHex4HexNAc5dHex3SP 2489 + +
NeuAc2Hex5HexNAc4dHex2 2513 + + + + + + +
NeuAcHex5HexNAc4dHex4 2514 + +
NeuAcHex6HexNAc5dHexSP and/or 2521 + + + +
NeuAc3 Hex2H exNAc5d Hex2
NeuAc2Hex6HexNAc4dHex and/or 2529 + + + +
NeuGcNeuAcHex5HexNAcAdHex2
NeuGc2Hex5HexNAcAdHex2 and/or 2545 + + +
NeuGcNeuAcHex6HexNAcAdHex
NeuAcHex5HexNAc5dHex3 2571 + + + + + + + +
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NeuAcHex6HexNAc5dHex2 2587 + + + + + + + + + + + +
NeuAcHex7HexNAc5dHex and/or 2603 + + + + + + +
NeuGcHex6HexNAc5dHex2
NeuGcHex8HexNAc5 and/or 2635 + +
NeuAcHex4HexNAc5dHex4SP
NeuAc2Hex5HexNAc4dHex3 2659 + +
NeuGcNeuAc2Hex5HexNAc4dHex 2674 + +
and/or NeuAc3Hex6HexNAc4
NeuAc2Hex4HexNAc5dHex2SP2 2714 + + + +
NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAc3Hex5HexNAc5
NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAcHex6HexNAc5dHex3 2733 + + + + + + + + + + + + +
NeuAcHex6HexNAc6dHex2 2791 + + + +
Hex6HexNAc6dHex3SP2 2805 +
NeuAcHex6HexNAc5dHex3SP 2813 +
NeuAc3Hex6HexNAc4dHex and/or 2820 +
NeuGcNeuAc2Hex5HexNAc4dHex2
NeuAc2Hex6HexNAc5dHex2 2879 + + + + + + + + + + + + +
NeuAcHex6HexNAc5dHex4 2880 + + + + +
NeuAc2Hex7HexNAc5dHex and/or 2895 + +
NeuGcNeuAcHex6HexNAc5dHex2
NeuAc3Hex6HexNAc4dHexSP and/or 2900 +
NeuGcNeuAc2Hex5HexNAc4dHex2SP
NeuGc2Hex6HexNAc5dHex2 2911 +
NeuAc2Hex5HexNAc6dHex2 2920 +
NeuAc2Hex6HexNAc6dHex and/or 2936 + + + + + + +
NeuGcNeuAcHex5HexNAc6dHex2
NeuAcHex6HexNAc6dHex3 2937 + +
NeuGc2NeuAcHex5HexNAc6 and/or 2951 +
NeuAc3 Hex5H exNAc4d Hex3
NeuAcHex7HexNAc6dHex2 2953 + + + + + + + +
NeuAc2Hex4HexNAc7dHex2 2961 +
NeuAc2Hex6HexNAc5dHex3 3025 + + + + + + + + + + +
NeuAc2Hex6HexNAc6dHex2 3082 +
NeuAcHex7HexNAc6dHex3 3099 + + + + + + + + + + + +
NeuAc2Hex6HexNAc5dHex3SP 3105 + +
NeuAc3Hex6HexNAc5dHex2 3170 + +
NeuAc2Hex6HexNAc5dHex4 3171 + + + + + +
NeuAc2Hex6HexNAc6dHex3 3228 +
NeuAc2Hex7HexNAc6dHex2 3244 + + + + +
NeuAcHex7HexNAc6dHex4 3245 + + + + + +
NeuAcHex7HexNAc7dHex3 3302 +
NeuAcHex8HexNAc7dHex2 3318 + + +
NeuAc2Hex7HexNAc6dHex3 3390 + + + + + + + + + +
NeuAcHex7HexNAc6dHex5 and/or 3391 + + +
NeuAcHex9HexNAc8
NeuAcHex8HexNAc7dHex3 3464 + + + + + +
NeuAc2Hex7HexNAc6dHex4 3536 + + + + + +
NeuAc2Hex8HexNac7dHex2 3609 + + +
NeuAcHex8HexNac7dHex4 3610 + + + +
NeuAc3Hex7HexNAc6dHex3 3681 + + + + + + +
NeuAcHex9HexNAc8dHex2 3683 + + +
NeuAc2Hex8HexNAc7dHex3 3755 + + + + + +
NeuAcHexlOHexNAc9 and/or 3756 + + + +
NeuAcHex8HexNAc7dHex5
NeuAc3Hex7HexNAc6dHex4 3827 + +
NeuAcHex9HexNAc8dHex3 3829 + + + +
NeuAc2Hex8HexNAc7dHex4 3901 + + +
NeuAc2Hex9HexNAc8dHex2 3974 + +
NeuAcHex9HexNAc8dHex4 3975 + +
NeuAc3Hex8HexNAc7dHex3 4046 + +
NeuAc2HexlOHexNAc9 and/or 4047 + +
NeuAc2 HexBH exNAc7d Hex5
NeuAc2Hex9HexNAc8dHex3 4120 +
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HexNAc>Hex_2
(terminal HexNAc, N>H) co
LU
w N L E g ~ O] a m ~ G J U
m U U V U
Proposed composition mlz
NeuAcHex3HexNAc4 1606 +
NeuAcHex3HexNAc4dHex 1752 +
NeuAcHex3HexNac5 1809 +
NeuGcHex3HexNAc5 1825 + +
Hex4HexNAc5dHexSP 1906 + +
NeuAcHex4HexNAc5 1971 + + + + + + +
Hex7HexNAc4SP and/or
Hex4HexNAc6SP2 and/or 2043 +
NeuAc2Hex3HexNAc4dHex
NeuAcHex4HexNAc5SP 2051 + + + + +
Hex4HexNAc5dHex2SP 2052 + + + +
NeuAcHex3HexNAc5dHex2 and/or 2101 +
NeuAc2Hex4HexNAc4Ac
NeuAcHex4HexNAc5dHex 2117 + + + + + + + + +
Hex4HexNAc5dHex2SP2 2132 +
Hex6HexNAc4dHex2SP and/or 2173 +
Hex3HexNAc6dHex2SP2
NeuAcHex4HexNAc6 2174 + + + + + +
NeuAc3Hex3HexNAc4 and/or
NeuGcHex6HexNAc4SP and/or 2188 + +
NeuAc2NeuGcHex2HexNAc4dHex
NeuAc2Hex3HexNAc4dHex2 and/or
Hex7HexNAc4dHexSP and/or 2189 + +
Hex4HexNAc6dHexSP2
NeuAc2Hex3HexNAc5dHex and/or 2246 + + + +
Hex7HexNAc5SP
NeuAc2Hex4HexNAc5 2262 +
NeuAcHex4HexNAc5dHex2 and/or 2263 + + +
NeuAc2Hex5HexNAc4Ac
Hex6HexNAc4dHex3SP and/or 2319 + + +
NeuGcNeuAcHex3HexNAc6
NeuAcHex4HexNAc6dHex 2320 + +
Hex7HexNAc4dHex2SP and/or 2335 + +
Hex4HexNAc6dHex2SP2
NeuAcHex5HexNAc6 2336 + +
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + +
Hex7HexNAc5dHexSP
NeuAcHex3HexNAc5dHex4 2393 +
NeuAcHex4HexNAc6dHexSP and/or
NeuGcHex6HexNAc4dHex2 and/or 2400 +
NeuAcHex7HexNAc4dHex
NeuAc2Hex4HexNAc5dHex 2408 + + +
NeuAcHex4HexNAc5dHex3 and/or 2409 + +
NeuAc2Hex5HexNAc4dHexAc
NeuAcHex5HexNAc6dHex 2482 +
NeuAcHex4HexNAc5dHex3SP 2489 + +
Hex6HexNAc7SP 2490 +
NeuAcHex6HexNAc5dHexSP and/or 2521 + + + +
NeuAc3 Hex2H exNAc5d Hex2
NeuAc2Hex5HexNAc6 2627 +
NeuGcHex8HexNAc5 and/or 2635 + +
NeuAcHex4HexNAc5dHex4SP
NeuAc2Hex4HexNAc5dHex2SP2 2714 + + + +
NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAc3Hex5HexNAc5
NeuGcNeuAc2Hex5HexNAc6 2935 +
NeuGc2NeuAcHex5HexNAc6 and/or 2951 +
NeuAc3 Hex5H exNAc4d Hex3
NeuAc2Hex4HexNAc7dHex2 2961 + P
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HexNAc=Hex_5
(terminal HexNAc, N=H) co m M u LL m U z ~ M Z_
LU
w N L E g O] a m ~ G J U
m U U V U
Proposed composition mlz
Hex5HexNAc5SP2 2002 + + + + + + +
NeuAcHex5HexNAc5 2133 + + + + + + + + + +
NeuAcHex5HexNAc5dHex 2279 + + + + + + + + + + + + + +
NeuAc2Hex5HexNAc5 2424 + + + + +
NeuAcHex5HexNAc5dHex2 2425 + + + + + + + + + +
NeuAc2Hex5HexNAc5dHex 2570 + + + + + + + +
NeuAcHex5HexNAc5dHex3 2571 + + + + + + + +
NeuAcHex6HexNAc6dHex 2644 + + + + + + + + + +
NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAc3Hex5HexNAc5
NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAcHex6HexNAc6dHex2 2791 + + + +
Hex6HexNAc6dHex3SP2 2805 +
NeuAc2Hex6HexNAc6dHex and/or 2936 + + + + + + +
NeuGcNeuAcHex5HexNAc6dHex2
NeuAcHex6HexNAc6dHex3 2937 + +
NeuAcHex7HexNAc7dHex 3010 + + +
NeuAc3Hex6HexNAc6dHex 3227 + +
NeuAc2Hex6HexNAc6dHex3 3228 +
NeuAc2Hex7HexNAc7dHex 3301 +
NeuAcHex7HexNAc7dHex3 3302 +
SP>1
U U U + +
(including sulphated and/or cn m M W w m U ~ M `" z
phosphorylated glycans) t w ~ L e m a m m o ~
00 U U V U
Proposed composition m/z
Hex3HexNAc2SP 989 + + +
Hex3HexNAc2dHexSP 1135 + +
Hex4HexNAc2SP 1151 + + + + +
Hex3HexNAc3SP 1192 +
Hex5HexNAc2SP 1313 +
Hex3HexNAc3dHexSP 1338 +
Hex4HexNAc3SP 1354 + +
Hex6HexNAc2SP 1475 + + + + + + + +
Hex4HexNAc3dHexSP 1500 + + + + + + + + + +
Hex5HexNAc3SP 1516 + + +
Hex6HexNAc2SP2 1555 +
Hex4HexNAc4SP 1557 + + + +
NeuAcHex3HexNAc3SP2 1563 + +
Hex4HexNAc4SP2 and/or 1637 + + + + + + +
Hex7HexNAc2SP
Hex4HexNAc3dHex2SP 1646 + +
Hex5HexNAc3dHexSP 1662 +
Hex6HexNAc3SP 1678 + + + + + + + + + + +
Hex4HexNAc4dHexSP 1703 + + +
NeuAcHex3HexNAc3dHexSP2 1709 + +
Hex4HexNAc4SP3 and/or 1717 +
Hex7HexNAc2SP2
Hex5HexNAc4SP 1719 + + + + + +
Hex7HexNAc2dHexSP 1783 +
NeuAcHex4HexNAc3dHexSP 1791 + + + + + +
Hex5HexNAc4SP2 and/or 1799 + +
Hex8HexNAc2SP
Hex5HexNAc3dHex2SP 1808 +
NeuAc2Hex5HexNAc2 and/or 1815 +
NeuAc2Hex2HexNAc4SP
Hex5HexNAc4dHexSP 1865 + + + + + + + + + + +
Hex6HexNAc4SP 1881 +
Hex4HexNAc5dHexSP 1906 + +
NeuAcHex6HexNAc2dHexSP and/or 1912 +
NeuAcHex3HexNAc4dHexSP2
NeuAcHex4HexNAc4SP2 1928 + +
Hex8HexNAc3SP and/or
Hex5HexNAc5SP2 and/or 2002 + + + + + + + +
NeuAc2Hex4HexNAc3dHex
NeuAcHex5HexNAc4SP 2010 + +
Hex5HexNAc4dHex2SP 2011 +
NeuGcHex5HexNAc4SP 2026 +
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Hex6HexNAc4dHexSP 2027 + +
Hex7HexNAc4SP and/or
Hex4HexNAc6SP2 and/or 2043 +
NeuAc2Hex3HexNAc4dHex
NeuAcHex7HexNAc3 and/or 2051 + + + + + + +
NeuAcHex4HexNAc5SP
Hex4HexNAc5dHex2SP 2052 + + + +
NeuAcHex4HexNAc4dHexSP2 2074 + +
NeuAc2Hex4HexNAc3dHexSP and/or
Hex8HexNAc3SP2 and/or 2082 + + +
Hex5HexNAc5SP3
NeuAcHex6HexNAc3dHexSP 2115 +
Hex7HexNAc3dHex2SP andlor
NeuAc2Hex3HexNAc3dHex3 and/or 2132 +
Hex4HexNAc5dHex2SP2
Hex8HexNAc3dHexSP and/or 2148 +
NeuAc2 Hex4H exNAc3d Hex2
NeuAcHex5HexNAc4dHexSP and/or 2156 + + + + + + +
NeuAcHex8HexNAc2dHex
Hex5HexNAc4dHex3SP 2157 +
NeuAc2Hex5HexNAc3dHex and/or 2164 + + +
Hex6HexNAc5SP2
NeuAc2Hex4HexNAc4SP2 2219 +
Hex6HexNAc5dHexSP 2230 + + + +
NeuAc2Hex3HexNAc5dHex and/or 2246 + + + +
Hex7HexNAc5SP
NeuAc2Hex4HexNAc4dHexSP and/or 2285 +
Hex11HexNAc2SP
NeuAcHex8HexNAc3SP and/or 2293 +
NeuAc3Hex4HexNAc3dHex
NeuAc2Hex5HexNAc4SP 2301 +
NeuAcHex5HexNAc4dHex2SP 2302 +
Hex6HexNAc4dHex3SP 2319 +
Hex7HexNAc4dHex2SP and/or 2335 + +
Hex4HexNAc6dHex2SP2
NeuAc2Hex4HexNAc4dHexSP 2365 + + +
NeuAc3Hex5HexNAc3SP and/or 2389 +
NeuAc2Hex5HexNAc4Ac4
NeuAc2Hex5HexNAc3dHex2SP 2390 + + + + + + + + +
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + +
Hex7HexNAc5dHexSP
NeuAcHex4HexNAc6dHexSP and/or
NeuGcHex6HexNAc4dHex2 and/or 2400 +
NeuAcHex7HexNAc4dHex
NeuAc2Hex6HexNAc3dHexSP 2406 + + +
NeuAcHex8HexNAc3dHexSP and/or 2439 +
NeuAc3 Hex4H exNAc3d Hex2
NeuAc2Hex5HexNAc4dHexSP and/or
NeuAc2Hex8HexNAc2dHex and/or 2447 + + + + + + +
Hexl2HexNAc2SP
NeuAcHex5HexNAc4dHex3SP and/or 2448 + + + + +
NeuAcHex8HexNAc2dHex3
NeuAcHex7HexNAc3dHex3 and/or 2489 + +
NeuAcHex4HexNAc5dHex3SP
Hex6HexNAc7SP 2490 +
NeuAcHex6HexNAc5dHexSP and/or
NeuAcHex9HexNAc3dHex and/or 2521 + + + +
NeuAc3 Hex2H exNAc5d Hex2
Hex6HexNAc5dHex3SP 2522 + +
Hex7HexNAc6dHexSP 2595 +
NeuGcHex8HexNAc5 and/or 2635 + +
NeuAcHex4HexNAc5dHex4SP
NeuAc2Hex4HexNAc5dHex2SP2 2714 + + + +
NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAc3Hex5HexNAc5
NeuAc3Hex5HexNAc4dHex2 and/or 2804 + +
NeuAcHex6HexNAc6dHexSP2
Hex6HexNAc6dHex3SP2 2805 +
NeuAc2Hex6HexNAc5dHexSP 2812 + + + + +
NeuAcHex6HexNAc5dHex3SP 2813 +
NeuAc3Hex6HexNAc4dHexSP and/or 2900 +
NeuGcNeuAc2Hex5HexNAc4dHex2SP
NeuAc3Hex6HexNAc5dHexSP 3104 + +
NeuAc2Hex6HexNAc5dHex3SP 3105 + +
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Table 7. Characteristic N-glycan signals of hESC. The 15 characteristic
neutral (uppeN panel)
and sialylated (lowerpanel) N-glycan signals of the hESC N-glycome. The
signals are expressed in
all the analyzed hESC samples and they are listed in order of relative
abundance (No) in each N-
glycan fraction. H. hexose, N. N-acetylhexosamine, F. deoxyhexose, S: N-
acetylneuraminic acid,
G: N-glycolylneuraminic acid. The proposed structural classification is
according to Fig. 3A and as
described in the text.
Neutral N-glycans:
No. m/z Proposed Proposed classification
[M+Na]+ composition
1. 1905.6 H9N2 high-mannose
2. 1419.5 H6N2 hi h-mannose
3. 1743.6 H8N2 hi h-mannose
4. 1257.4 H5N2 hi h-mannose
5. 1581.5 H7N2 hi h-mannose
6. 1079.4 H3N2F1 low-mannose
7. 2067.7 H10N2 other types lucos lated
8. 1095.4 H4N2 low-mannose
9. 933.3 H3N2 low-mannose
10. 1663.6 H5N4 complex-type
11. 1622.6 H6N3 h brid/monoantenna
12. 1809.6 H5N4F1 complex-type
13. 1460.5 H5N3 h brid/monoantenna
14. 1485.5 H3N4F1 complex-type; terminal N-acetylhexosamine (N>H)
15. 1444.5 H4N3F1 h brid/monoantenna
Sialylated N-glycans:
m/z Proposed
No. [M-H]' composition Proposed classification
1. 2076.7 S1H5N4F1 complex-type
2. 2222.8 S1 H5N4F2 complex-type; complex fucosylation
3. 2367.8 S2H5N4F1 complex-type
4. 1930.7 S1H5N4 complex-type
5. 2441.9 S1H6N5F1 complex-type
6. 2092.7 G1H5N4F1 complex-type
7. 2117.8 S1H4N5F1 complex-type; terminal N-acetylhexosamine (N>H)
8. 2587.9 S1 H6N5F2 complex-type; complex fucosylation
9. 2368.9 S1 H5N4F3 complex-type; complex fucosylation
10. 2263.8 S1H4N5F2 complex-type; complex fucosylation;
terminal N-acet Ihexosamine N>H
11. 1711.6 S1H4N3F1 h brid/monoantenna
12. 2279.8 S1H5N5F1 complex-type; terminal N-acetylhexosamine N=H2:5
13. 2238.8 G 1 H5N4F2 com lex-t e; complex fucosylation
14. 2733.0 S2H6N5F1 complex-type
15. 2807.0 S1H7N6F1 com lex-t e
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Table 8. NMR analysis of the major neutral N-glycans of hESC. The identified
signals were
consistent with high-mannose type N-glycan structures such as the structures A-
D that have
monosaccharide compositions H7_9N2. The significant signals in the NMR
spectrum can be
explained by the following glycan structure combinations: A+B+C+D, A+B+D,
A+C+D, B+C+D,
A+D, or B+C. Reference data is after Fu et al. (Fu, D., et al., 1994,
Carbohydr. Res. 261, 173-186)
and Hard et al. (Hard, K., et al., 1991, Glycoconj. J. 8, 17-28).
Monosaccharide symbols are as in
Supplementary Figure S 1.
A B C D
TO ~ TO ~ TO ~ ~
TO Glycan residue 1H-
NMR chemical shift (ppm)
Residue Linkage Proton A B C D hESC')
H-1a 5.191 5.187 5.187 5.188 5.188
D-GIcNAc H-113 4.690 4.693 4.693 4.695 4.694
NAc 2.042 2.037 2.037 2.038 2.038
(3-D-GIcNAc 4 H-1 4.596 4.586 4.586 4.600 4.596
NAc 2.072 2.063 2.063 2.064 2.061
(3-D-Man 4,4 H-1 4.775 4.771 4.771 4.780 2)
H-2 4.238 4.234 4.234 4.240 4.234
a-D-Man 6,4,4 H-1 4.869 4.870 4.870 4.870 4.869
H-2 4.149 4.149 4.149 4.150 4.153
a-D-Man 6,6,4,4 H-1 5.153 5.151 5.151 5.143 5.148
H-2 4.025 4.021 4.021 4.020 4.023
a-D-Man 2,6,6,4,4 H-1 5.047 5.042 5.042 5.041 5.042
H-2 4.074 4.069 4.069 4.070 4.069
a-D-Man 3,6,4,4 H-1 5.414 5.085 5.415 5.092 5.408 / 5.085
H-2 4.108 4.069 4.099 4.070 4.102 / 4.069
a-D-Man 2,3,6,4,4 H-1 5.047 - 5.042 - 5.042
H-2 4.074 - 4.069 - 4.069
a-D-Man 3,4,4 H-1 5.343 5.341 5.341 5.345 5.346 / 5.338
H-2 4.108 4.099 4.099 4.120 4.102
a-D-Man 2,3,4,4 H-1 5.317 5.309 5.050 5.055 5.310 / 5.057
H-2 4.108 4.099 4.069 4.070 4.102 / 4.069
a-D-Man 2,2,3,4,4 H-1 5.047 5.042 - - 5.042
H-2 4.074 4.069 - - 4.069
1) Chemical shifts determined from the center of the signal.
2) Signal under HDO.
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Table 9. NMR analysis of the major sialylated N-glycan core structures of
hESC. The
identified signals were consistent with sialylated biantennary complex-type N-
glycan structures
such as the structures A-D that have monosaccharide compositions Si_2H5N4f'o-
i. Reference data is
after Hard et al. (Hard, K., et al., 1992, Eur. J. Biochem. 209, 895-915) and
Helin et al. (Helin, J., et
al., 1995, Carbohydr. Res. 266, 191-209). The significant signals in the NMR
spectrum can be
explained by the structural components of these reference structures (not
shown). Monosaccharide
symbols are as in Supplementary Figure Sl.
A B C D
a3 9.;:) 6 a6
p4 p4 R2 R2 R2 R2 3 6 6 3 a6 R4 4 R4 R4
R4 4 R4 R4
a6
Glycan residue 'H-NMR chemical shift (ppm)
Residue Linkage Proton A B C D hESC
D-GIcNAc H-1a 5.188 5.189 5.181 5.189 5.182 / 5.188
NAc 2.038 2.038 2.039 2.038 2.038
H-la - - 4.892 - 4.893
a-L-Fuc 6 H-1(3 - - 4.900 - 4.893
CH3a - - 1.211 - 1.210
CH3(3 - - 1.223 - 1.219
(3-D-GIcNAc 4 H-lp 4.604 4.606 n.a. 4.604 4.605
NAc 2.081 2.081 2.096 2.084 2.081 / 2.095
(3-D-Man 4,4 H-1 n.a. n.a. n.a. n.a. n.a.
H-2 4.246 4.253 4.248 4.258 4.256
a-D-Man 6,4,4 H-1 4.928 4.930 4.922 4.948 4.927
H-2 4.11 4.112 4.11 4.117 n.a.
(3-D-GIcNAc 2,6,4,4 H-1 4.581 4.582 4.573 4.604 4.579 / 4.605
NAc 2.047 2.047 2.043 2.066 2.047 / 2.069
H-1 4.473 4.473 4.550 4.447 4.447 / 4.472
(3-D-Gal 4,2,6,4,4 / 4.545
H-4 n.a. n.a. n.a. n.a. 4.185
a-D-Man 3,4,4 H-1 5.118 5.135 5.116 5.133 5.118 / 5.134
H-2 4.190 4.196 4.189 4.197 4.195
(3-D-GIcNAc 2,3,4,4 H-1 4.573 4.606 4.573 4.604 4.579 / 4.605
NAc 2.047 2.069 2.048 2.070 2.047 / 2.069
(3-D-GaI 4,2,3,4,4 H-1 4.545 4.445 4.544 4.443 4.445 / 4.545
H-3 4.113 n.a. 4.113 n.a. n.a.
1) Chemical shifts determined from the center of the signal.
n.a.: Not assigned.
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Table 10. Relative proportions (%) of sialylated N-glycan signals in hESC and
differentiated cell lines.
N N O p O! 4m
Proposed N N N N N N ~~ M M N N N
m/Z V) M N M N M V~ M
composition w m w m ~: w m r w m r
LL W f/) LL W f/) LL W fn LL W U)
S1H4N3F1 1711 2,16 2,68 2,73 2,25 3,02 3,46 1,77 3,16 3,05 1,86 2,41 2,89
S1H6N3 1889 1,44 2,17 3,05 0,00 1,64 2,53 1,74 2,18 2,45 0,96 2,59 0,93
S1H5N3 1727 1,54 1,48 1,86 0,00 1,36 3,15 0,99 1,06 1,71 1,07 2,39 0,79
S1H4N3 1565 1,13 1,13 1,19 0,00 1,27 1,52 0,93 0,99 1,50 0,76 0,69 0,00
S1H5N3F1 1873 0,81 2,26 3,13 0,00 1,46 2,14 1,42 1,68 1,86 0,00 2,17 1,31
S2H5N3F1 2164 0,00 0,61 1,64 0,00 0,59 0,00 0,00 0,56 0,00 0,96 0,00 0,00
S1H6N3F1 2035 0,00 1,28 1,23 0,00 0,66 2,05 0,00 0,71 1,08 0,00 0,66 0,71
S1H5N4F1 2076 28,66 28,27 18,93 26,02 30,38 15,78 27,66 25,28 26,15 25,91
23,90 21,83
S1H5N4F2 2222 12,84 3,35 3,98 15,53 2,83 2,19 10,12 5,19 2,62 9,18 3,21 1,61
S2H5N4F1 2367 5,89 4,52 2,88 9,69 3,74 2,40 7,73 4,22 3,55 7,22 4,95 7,08
S1H5N4 1930 5,55 5,53 5,03 4,30 4,91 3,37 6,13 4,70 5,57 6,18 4,89 3,76
S1H6N5F1 2441 5,06 3,13 3,70 5,85 3,86 4,13 3,97 4,28 4,39 4,07 3,31 4,82
G1H5N4F1 2092 3,61 3,10 0,00 2,81 2,56 0,00 5,00 2,85 0,00 4,87 1,89 0,00
S1H4N5F1 2117 3,69 5,33 3,62 3,27 4,17 4,20 2,27 4,64 3,14 2,12 4,74 4,81
S1H6N5F2 2587 2,67 0,70 1,51 4,06 0,66 0,00 1,95 1,07 1,28 2,25 1,13 1,09
S1H5N4F3 2368 1,91 1,62 1,08 3,57 1,01 0,13 1,14 0,73 1,47 3,16 2,81 0,82
S1H4N5F2 2263 4,17 1,33 1,27 2,44 1,00 2,91 1,24 2,15 0,98 1,72 1,35 1,08
S1H5N5F1 2279 1,96 7,31 11,76 2,38 12,21 13,72 1,53 7,97 11,61 1,73 9,91 14,65
S2H6N5F1 2732 1,56 0,82 1,36 2,18 0,80 0,00 1,16 0,35 1,25 1,46 0,28 2,21
S1H6N4F1 2238 1,44 1,06 1,69 2,82 0,79 1,46 1,56 2,57 2,00 0,00 0,69 1,02
S1G1H5N4 2237 1,05 0,56 0,00 0,00 0,77 0,00 2,23 1,12 0,00 2,22 1,66 0,00
S1H7N6F1 2807 1,42 0,47 0,00 2,26 0,47 1,23 0,70 0,95 1,86 1,03 1,13 1,70
S1H7N6F3 3099 0,68 0,00 0,00 1,98 0,00 0,00 0,45 0,06 0,57 1,84 0,00 0,00
S2H4N5F1 2408 1,72 0,77 0,00 2,23 0,43 0,00 0,00 0,72 0,00 0,94 0,00 0,00
S1H5N5F2 2425 1,00 1,60 1,78 2,01 1,20 2,09 0,83 1,90 1,85 1,04 1,77 1,59
S2H5N4 2221 0,00 1,48 0,00 0,08 1,42 1,31 2,14 1,70 1,39 2,62 2,13 4,35
G2H5N4 2253 0,00 0,00 0,00 0,00 0,52 0,00 2,37 1,13 0,00 2,01 0,28 0,00
G1H5N4 1946 1,21 1,28 0,00 0,00 0,00 0,00 1,28 0,57 0,00 1,68 0,00 0,00
S1H6N4F2 2384 0,00 0,93 1,13 0,00 0,31 0,00 2,64 0,91 0,00 1,34 0,00 0,00
S1H6N5 2295 1,26 1,03 1,73 0,00 1,22 0,00 1,21 1,00 0,69 1,10 1,09 0,00
S1H6N5F3 2733 0,66 0,57 0,00 1,80 0,08 2,12 1,03 0,78 1,03 0,00 1,69 0,00
S2H6N4 2383 1,13 1,04 0,00 0,00 0,47 0,00 0,00 0,14 0,00 1,76 0,00 0,00
S1H7N6F2 2953 0,77 0,00 0,00 0,83 0,00 0,00 0,00 0,00 0,00 1,11 0,00 0,00
S1H8N7F1 3172 0,00 0,00 0,00 1,66 0,00 0,00 0,00 0,00 0,00 0,74 0,00 0,00
S1H4N4F1 1914 1,26 2,30 1,94 0,00 2,00 1,87 0,99 2,32 2,38 0,00 1,61 1,06
S3H6N5 2878 0,00 0,00 0,00 0,00 0,00 1,33 1,92 0,42 0,00 0,00 0,37 0,00
S1H6N4F1Ac 2280 0,72 1,86 2,86 0,00 3,05 5,74 0,00 0,72 1,93 0,72 2,23 3,35
S2H6N5F2 2879 0,00 0,00 0,00 0,00 0,48 0,00 0,00 0,47 0,00 1,11 0,53 0,00
S1H5N5 2133 0,00 0,84 1,81 0,00 1,22 2,68 0,00 0,44 1,78 0,81 1,24 0,73
S2H5N5F1 2570 0,00 0,79 1,74 0,00 0,76 0,00 0,00 0,12 0,49 0,72 1,55 2,04
S2H7N6F1 3098 0,00 0,00 0,00 0,00 0,00 0,00 0,67 0,04 0,00 0,00 0,09 1,66
S1H6N6F1 2644 0,00 0,64 1,92 0,00 0,88 2,27 0,00 1,21 2,37 0,00 1,29 3,00
S1H5N6F2 2482 0,00 1,20 1,86 0,00 0,00 1,92 0,00 0,57 1,54 0,00 0,54 1,20
S1H7N5F1Ac 2645 0,00 0,00 0,98 0,00 0,56 2,02 0,00 0,55 0,56 0,00 0,92 2,12
S1H5N5F3 2571 0,00 0,23 0,00 0,00 0,23 0,00 0,00 0,68 1,50 0,00 0,91 1,26
S1H4N4 1768 0,00 0,55 1,17 0,00 0,46 0,00 0,00 0,17 0,00 0,00 0,32 0,00
S2H2N3F1 1678 1,04 2,17 3,95 0,00 1,87 4,08 0,94 2,12 2,86 0,89 2,58 1,69
S2H4N3F1 2002 0,00 1,26 2,86 0,00 1,03 2,35 1,27 1,62 0,95 0,00 1,58 0,99
S2H3N3F1 1840 0,00 0,78 1,42 0,00 0,58 1,92 1,01 0,55 0,00 0,00 0,51 0,97
S2H4N2F1 1799 0,00 0,00 1,22 0,00 0,43 1,92 0,00 0,07 0,60 0,00 0,00 0,89
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Table 11. Relative proportions (%) of neutral N-glycan signals in hESC and
differentiated cell lines.
Proposed m/Z N M y m y m co M
composition LLM W M (n M
LL N W N f/~ N LL N LU N N N LL N W N V~ N
H9N2 1905 19,19 14,65 17,06 18,69 15,98 15,26 19,92 1,07 0,00 18,96 0,00 0,00
H8N2 1743 21,08 14,38 16,76 14,51 15,32 16,45 20,67 0,87 0,87 21,12 1,56 1,04
H6N2 1419 18,41 18,31 14,47 16,18 17,95 16,33 16,74 1,66 2,13 16,35 2,51 1,22
H7N2 1581 13,01 11,25 10,79 10,10 10,86 11,15 12,27 1,76 1,62 12,17 2,44 1,47
H5N2 1257 9,75 14,50 11,50 10,71 14,37 11,51 8,13 3,10 3,87 8,27 3,78 2,33
H3N2F1 1079 1,19 3,78 4,20 3,37 2,97 4,64 0,95 2,62 2,39 1,12 3,01 2,31
H4N2 1095 2,07 2,87 2,80 2,56 2,84 2,36 1,63 0,35 0,43 1,43 0,78 0,78
H1ON2 2067 2,82 1,81 1,87 2,79 2,05 1,76 2,25 0,38 0,33 2,14 0,43 2,29
N2N2F1 917 0,56 2,34 2,82 1,23 1,67 3,62 0,35 0,43 0,43 0,47 0,60 0,24
H3N2 933 1,10 2,20 2,30 2,08 1,82 2,12 0,74 13,30 12,32 0,61 11,22 8,25
H2N2 771 0,43 1,07 1,97 0,77 0,73 1,96 0,00 0,65 1,04 0,00 0,81 1,11
H1N2 609 0,00 0,00 0,00 0,56 0,00 0,00 2,90 0,65 0,42 3,99 0,53 0,36
H5N2F1 1403 0,32 0,44 0,41 0,27 0,40 0,57 0,00 0,00 0,22 0,00 0,31 0,35
H4N2F1 1241 0,26 0,46 0,42 0,36 0,46 0,35 0,21 0,07 0,30 0,14 0,30 0,30
H6N2F1 1565 0,00 0,14 0,17 0,00 0,21 0,42 0,00 0,53 0,55 0,00 0,56 0,34
H11N2 2229 0,00 0,10 0,12 0,24 0,00 0,00 0,10 16,44 16,44 0,07 17,49 12,47
H6N3 1622 0,57 0,86 0,97 1,51 0,96 0,91 0,58 0,64 0,56 0,53 0,84 0,69
H5N3 1460 0,50 0,58 0,87 1,27 0,70 0,61 0,51 1,11 0,96 0,55 0,72 0,84
H3N3F1 1282 0,33 0,48 0,78 0,59 0,48 0,54 0,35 0,85 1,06 0,41 0,40 0,68
H4N3F1 1444 0,55 0,46 0,44 0,77 0,66 0,49 0,73 0,08 0,22 0,65 0,28 0,33
H3N3 1136 0,28 0,28 0,78 0,64 0,43 0,39 0,31 0,08 0,27 0,33 0,05 0,03
H4N3 1298 0,59 0,45 0,74 0,80 0,63 0,52 0,45 0,22 0,23 0,50 0,13 0,06
H5N3F1 1606 0,28 0,34 0,30 0,74 0,32 0,20 0,23 10,77 10,69 0,11 11,14 9,82
H2N3F1 1120 0,00 0,35 0,66 0,00 0,33 0,41 0,00 0,06 0,00 0,00 0,08 0,11
H6N3F1 1768 0,33 0,32 0,14 0,39 0,21 0,29 0,00 0,61 0,68 0,00 0,08 0,25
H4N3F2 1590 0,00 0,17 0,15 0,00 0,24 0,00 0,00 1,76 1,17 0,17 0,97 1,23
H5N4 1663 2,29 1,89 1,14 1,78 1,82 0,91 2,19 0,63 0,52 2,75 0,12 0,28
H5N4F1 1809 1,33 1,27 0,57 1,50 1,37 0,66 3,86 1,91 2,07 3,69 1,30 2,68
H3N4F1 1485 0,41 0,47 0,67 1,03 0,64 0,77 0,57 0,31 0,46 0,55 0,06 0,17
H5N5 1866 0,00 0,11 0,43 1,33 0,32 0,55 0,00 0,81 0,82 0,00 0,06 0,28
H4N4F1 1647 0,32 0,40 0,34 0,52 0,40 0,40 0,46 14,86 15,30 0,38 14,82 17,75
H5N4F2 1955 0,42 0,26 0,18 0,00 0,38 0,31 0,83 0,23 0,16 0,89 0,04 0,40
H4N5 1704 0,00 0,00 0,27 1,35 0,07 0,33 0,00 0,09 0,00 0,00 0,33 0,38
H6N5F1 2174 0,36 0,27 0,11 0,21 0,22 0,00 0,73 2,07 1,13 0,50 1,03 1,09
H5N4F3 2101 0,21 0,22 0,14 0,00 0,27 0,21 0,47 0,11 0,34 0,47 0,02 0,29
H4N5F1 1850 0,00 0,20 0,21 0,28 0,25 0,32 0,00 0,48 0,41 0,00 0,07 0,36
H6N5 2028 0,34 0,19 0,12 0,27 0,25 0,00 0,56 0,89 1,01 0,30 0,19 0,60
H3N5F1 1688 0,00 0,21 0,29 0,00 0,19 0,35 0,18 14,28 15,44 0,14 16,85 22,44
H4N4 1501 0,02 0,27 0,40 0,18 0,08 0,36 0,00 0,30 0,00 0,00 0,10 0,36
H4N5F2 1996 0,00 0,23 0,14 0,00 0,23 0,31 0,15 0,06 0,00 0,00 0,20 0,40
H3N4 1339 0,00 0,34 0,52 0,00 0,00 0,23 0,00 0,22 0,25 0,00 0,27 0,33
H4N4F2 1793 0,00 0,22 0,16 0,00 0,23 0,30 0,00 0,19 0,12 0,14 0,04 0,10
H6N4 1825 0,00 0,07 0,32 0,10 0,00 0,37 0,00 0,16 0,10 0,00 0,04 0,10
H4N5F3 2142 0,50 0,11 0,06 0,00 0,00 0,00 0,00 1,65 2,00 0,10 2,22 2,25
H5N6F2 2361 0,00 0,14 0,00 0,00 0,12 0,00 0,00 0,21 0,00 0,00 0,31 0,13
H5N5F3 2304 0,00 0,15 0,16 0,00 0,17 0,31 0,00 0,11 0,00 0,00 0,43 0,03
H5N5F1 2012 0,00 0,12 0,12 0,27 0,12 0,00 0,00 0,19 0,14 0,00 0,06 0,09
H7N4 1987 0,00 0,07 0,11 0,00 0,00 0,00 0,00 0,09 0,13 0,00 0,03 0,09
H3N5 1542 0,00 0,21 0,00 0,05 0,00 0,13 0,00 0,09 0,17 0,00 0,04 0,10
H2N4F1 1323 0,19 0,00 0,08 0,00 0,30 0,33 0,00 0,00 0,21 0,00 0,38 0,42
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Table 12.Proposed structures for acidic N-glycan signals in hESC or
differentiated cells,symbols Table13.
m/z structure m/z structure m/z structure
SP
~ ~ sN
1151 1719 1906
SP
A
1338 1727 1914
~~~ SP ~ ~ E
1354 1744 1930
1362 1752 1946
1403 1760 1947
)WE
1475 1768 1971
SP SP
A
1500 1791 2002
1516 1799 2003
~- SP SP 5P
1541 ~ 1808 A 2010
A SP .~` IAV: SP
1549 1824
2011
SP
1557 1831 2018
SP
Al
1565 1840 2027
e'- SP SP
\ 0.1~~~
1637 1849 2035 Vi
SP SP
1678 1865 2051
SP sP
A ,
1703 1873 v 2052
WE
7 SP
Alll-
A
1711 1889 2068
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mlz structure m/z structure m/z structure
-11 me",
2076 2246 2391
2082 2253 2400
~-D3,:"
2092 ~~ 2254 2408
g, A
2117 2263 2425
-'o
2133 2279 2433
2156 SP 2280 2441
BP SP
*
2157 2295 2447
SP Q_~`., ssps
2164 2302 2448 4A SP
~ SP
2174 2319 \A pA 2456
Wv
2178 2320 2457
A
2214 2321 2482 A
on`~~~
2221 2367 2483
2222 2368 2512 CD sp SP
2230 2376 2521
A
A
:SP
2237 2383 2522
~312238 2384 ltl 2528
.il
2239 2390 2529
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mlz structure m/z structure m/z structure
on
A
2544
2733
A 3025
2570 Bm
2791 3026
2571
2806
3098
2579 SP 2807 A
\~~fsw ~ 3099
-Wffil cJ ~.,3/6/a8 \'WE A sP
2586 2813
2587 2848 3170
41
W:JRU\
2603 2864 3172
A
2627 2878 ~ 3245 &
A
A
2644 2879 3317
A
w Q
2880 3390 C~6 ~
2645 Ac
:.r SP \~
~~- m I
~ * ~ ~
A In0 a
2660 2886 3463 9 A
sP W;? sp ~4
~ \Q
A
A 1a~ Q\
2668 2887 3608
0 14,
l0~ . ~
n
2683 SP ~~ 2936 3610
~ sP
A ~
~~. <:,
2714 2953 3682
~sP > ~\
2725 3024 3756 w
2732
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Table 13. Proposed stnietarec for neutral N-glycnn cienalc detected in hESC or
differentiated cellc.Svmholc Tablel4.
m/z Structure m/z structure m/z structure
568,19 -I O D 1209,44 1485,53
609,21 1216,4 1501,53
714,24 1225,43 1517,55
730,24 1241,43 1540,5
755,27 0 1257,42 1542,56
771,26 1266,46 1555
892,29 1282,45 1565,53 ',
D 10 0
901,33 1298,45 1581,53
917,32 1323,48 1590,57
933,31 1339,48 1606,56
1031,33 1378,45 :1622,56
1054,34 1393 1631,59
elm
1079,38 1403,48 1647,59
o
1095,37 1419,48 1663,58
1120,4 1444,51 ASMAS 1688,61 A An
1136,4 imm-* 0 1460,5 0 1702,56
S
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m/z structure m/z structure m/z structure
1704,61 1971,69 2149,74 ~ 1717 1980,73 2158,78
1720,63 1987,69 2174,77
1743,58 ~~ 1996,72 2183 81
,
1752,62 2012,72 2190,77
1768,61 im-om 2019,7 2199,8 A
1784,61 2021,76 2215,8
W -0 A
1793,64 2028,71 2229,74 ~WO
1809,64 2037,75 2231,79
1825,63 2041 2304,84
1850,67 2053,75 2320,83
1864,61 ~ 2067,69 2361,87
~~lt
1866,66 2101,76 2391,79
1882,68 2117,75 2393,85
1905,63 2126,79 2466,89 A
~ A
1914,67 2133,75
1955,7 2142,78
~
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Table 14. Lectin staining of human embryonic stem cells.The glycan structures
are presented in
colour symbols,given at the end of Table 19.The reducing end of the N-glycans
is on left for N-
glycans in Tablesl2 and 13, and on right in Tables 14-19 (mirror images to
ones in 12 and 13).The
linkages of N-glycans are indicated in NMR Tables 8 and 9, and in Tables 12-19
based on the
Consortium for Functional Glycomics, USA recommendations, 1-4 linkages
(Man(34,GlcNAc(34,Ga1(34,Gala4 on Lactosylresidue in globostructres,GalNAc(34
on on
Lactosylresidue in ganliostructures) are horizontal -, 1-6 linkages (Mana6,
NeuAc/sialic acida6,
G1cNAc(36) are \ in Tables 14-19, except Fuca6 above above reducing end G1cNAc
in, and / in
Tables 12 and 13, 1-3 linkages (Mana3,Fuca3,Neu5Ac/Neu5Gc/sialic
acida3,Ga1(33,G1cNAc(33,
GalNAca3GalNAc(33 and GalNAc(33 on Gala4 at non-reducing end of Forsman and
Globoside(Gb4)
and elongated globoseries glycolipid structures respectively) are / in Tables
14-19, and \ in Tables 12
and 13 (for N-glycan compatible structures. Fuca2 is indicated by vertical
line below Gal(33/Gal(34-
residue. SP in Tables 12 and 13 indicates sulphated or fosfate and is
preferably sulfate on compelx
type N-aglycans comprising N-acetyllactosamine residues and fosfate in
High/Low Mannose
glycans.In tables 14-19 S is sialic acid (preferably Neu5Ac and/or Neu5Gc), LN
is N-cetyl-
lactosamine, preferably Gal(34G1cNAc, LN type 1 is Gal(33G1cNAc, Lex is Lewis
x, Ley is Lewis y,
Leb is Lewis b. Regular abbreviations of plant leactins are used, these are
available e.g. from catalog
of EY Labs USA. MEF is mouse embryonic fibroblast feeder cell, FES indicates
embryonic stem cell
line and number specifies the line, EB is embryonic body.
Lectin epitope FES22 FES30 f29+30 MEF
PSA Mana - - +
LTA Lex -/+ - - +
UEA H type 2 + - zz+, 29-
MAA Sa2-3 + + + -
SNA
Sa2-6 `o (+/-l (+/-l +
RCA LN + + 1+ +
PNA Gal(31- Q-El + + + -
PWA polyLN (I) + + + +
STA polyLN (i) +
WFA C'7a1NAc [i ^ + + M + -
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Table 15. Antibody staining of human embryonic stem cells. Antibodies are
listed in Table
20.
Epitope FES22,29,30 MEF
~'?; -
G1oboH -/+ ~\ ~
::
H type 1 +
~~~..
Htype2 + -
Leb, Ley, +
Leb -/+ -
H type 2 ~ -/+ -
H type 2 ~ -/+ -
Ley ? ?
LN (1) + -
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Table 16. Antibody staining of human embryonic stem cells.
Epitope FES22,29,30 MEF
Forssman IIP O- - -/+
Low Man -/+ -
Globoside
LacdiNAc
GM3 + +
GM3 + +
Lex C>W
-? -?
sLex -? -?
sLea ~ - -
Table 17. FACS analysis (lectins) of human embryonic stem cells (% of positive
cells).
Lectin Epitope FES29 MEF FES30 staining
(MEF) (matrigel (FES30)
PNA Galpl-3Ga1NAc 0-0 80% 20% 84% +
PSA Mana 51% 64% 54% -
MAA Sa2-3 *P 27% 9% 33% +
PWA polyLN (I) CW~Dw 3% 11% 1% +
UEA H type 2 63% 2% 42% -
STA polyLN (i) PIIF 9% -
OMPM
MBL Mana 0%
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Table 18. FACS analysis (antibodies) of human embryonic stem cells (% of
positive cells).
epitope FES29 MEF FES30 Staining
(MEF) matrigel (FES22,29,30)
LN type 1 JM 87% +
SSEA-3 ~ 74% +
SSEA-4 ee~ 23% +
Tra.-1-60 Podocalyxin 47% 2% 22%
KS
Table 19. TLC blot of human embryonic stem cells. Experiments with low amounts
of
Sample, + indicates potential reactivity, - not done or need experiments,2
columns on right for
comparision. Monosacharide symbols below and with Table 14, reducing end on
the right.
epitope FES29 FES30 FES61 FACS Cell
Staining
(FES29) FES22,29,30
LN type 1 0~" - - - + +
asialo GM1 r,j^-~-~ + - -
SSEA-3 ~ J~ - - - + +
SSEA-4 - - + + +
Gal(31- ~^ - - -
Q r ., ,,.T A
asialo GM2 + - -
globoside - - - +/-
Forssman + + + -
H (1) - - - +
globo H - - - +/-
H (2) - - - +
Ley - - - ?
Leb - - - +/-
Lea p~ - - - -
Hex=Gal Hex=Glc WHexNAc=GaINAc ; NeuSAc
Hex=Man AdHex= Fuc 13 HexNAc=GIcNAc NeuSGc
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Table 20.
Code Producer code Clone Specificity host/isotype
GF 279 Abcam ab3352 K21 Lewis c, LacNAc (LN) Type 1 mouse/IgM
MAB-S301
GF 280 Glycotope (Nemod TF2) TF-antigen Gal 3GaINAc
MAB-S305
GF 281 Glycotope (A68-E/E3) TF-anti en Gal 3GaINAc Mouse I G1
GF 283 Acris DM3122 2-25LE Lewis b (Leb) mouse/IgG
GF 284 Acris DM3015 B393 H Type 2 H (2) mouse/IgM
GF 285 Acris DM3014 B389 H Type 2, Lewis b, Lewis mouse/I G1
GF 286 Acris BM258P BRIC 231 H Type 2, H (2) mouse/I G1
GF 287 Abcam ab3355 17-206 H Type 1 , H 1 mouse/IgG3
GF 288
GF403 Glycotope MAB-S206 A69-A/E8 Globo H mouse/IgM
GF 289 Glycotope MAB-S201 A70-C/C8 Lewis (Ley) mouse/IgM
GF 290 Glycotope MAB-S204 A51-B/A6 H type 2, H (2) mouse/I A
GF 304 Chemicon CBL205 PR5C5 Lewis a
GF 305 Chemicon CBL144 28 Lewis x (Lex)
GF 307 Chemicon MAB2096 KM93 Sialyl Lewis x (Slex)
GF 353 Chemicon MAB4303 MC-631 SSEA-3
GF 366 Abcam ab23949 polyclonal Gb4, globoside rabbit
GF 367 Acris SM1160P Gb3 globotriose
GF 368 Leiden University 259-2A1 LacdiNAc mouse/IgG3
GF 369 Leiden University 273-3F2 LacdiNAc mouse/IgM
GF 370 Leiden University 290-2E6 a3-fucosyl-LacdiNAc mouse/IgM
GF 371 Leiden University 291-3E9 a3-fucosyl-LacdiNAc
GF 372 Acris B35.1 Sial 1-Tn
GF 373 Acris DM3184P PN-15
GF 305 Chemicon CBL144 28 Lewis x (Lex)
GF 307 Chemicon MAB2096 KM93 Sialyl Lewis x (Slex)
GF 401 Acris BM4091 FOM-1 Forssman antigen rat/I M
low-mannose N-glycan (low
GF 402 Leiden University 100-4G11 man) mouse/IgG
GF 418 Alexis MBr1 Globo-H
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TABLE 21
~
Trivial name Terminal epitope W ~q M
N+
+~ +/-
LN type 1, Le' Ga1R3G1cNAc 0+
L++
Lea Gal 3 Fuca4 G1cNAc L+ +/- +/-
H type 1 Fuca2Ga1 3G1cNAc L++ +/- +/-
Leb Fuca2Ga1 3 Fuca4 G1cNAc + +/- +/-
sial 1 Lea SAa3Ga1 3 Fuca4 G1cNAc +/- +/-
a3'-sial 1 Le' SAa3Ga1 3G1cNAc
N++
LN type 2 Gal(34G1cNAc O++ + +
L+/-
N++
Le' Ga1(34(Fuca3)G1cNAc O+/- +/- +/-
L+/-
N+
H type 2 Fuca2Ga1(34G1cNAc O+/- +/- +/-
L+/-
Le'' Fuca2Ga1 4 Fuca3 G1cNAc + +/- +/-
sial 1 Le' SAa3Ga1 4 Fuca3 G1cNAc + +/- +/-
a3'-sialyl LN SAa3Ga1(34G1cNAc 0+ N+ N+
a6'-sialyl LN SAa6Ga1(34G1cNAc N+ N++ N++
+/- +/-
Core 1 Gal 3Ga1NAca 0+
H type 3 Fuca2Ga1(33GalNAca O+ +/- +/-
sialyl Core 1 SAa3Ga1(33Ga1NAca 0+
disialyl Core 1 SAa3Ga1(33(SAa6)GalNAca 0+
type 4 chain Gal 3Ga1NAc L+ +/- +/-
H type 4 Fuca2Ga1 3Ga1NAc L+ +/- +/-
a3'-sial 1 type 4 SAa3Ga1 3Ga1NAc L++ +/- +/-
LacdiNAc Ga1NAc 4G1cNAc N+ +/- +/-
Lac Gal 4Glc L+ q q
G1cNAc(3 G1cNAc(3 ~+ q q
Tn Ga1NAca q
sialyl Tn SA0GalNAca
Ga1NAc Ga1NAc L+ N+ q q
poly-LN, i repeats of Gal 4G1cNAc 3 + q q
poly-LN, I Gal 4G1cNAc 3 Gal 4G1cNAc 6 Gal L+ +/- +/-
1) Stem cell and differentiated cell types are abbreviated as in other parts
of the present document; st.3
indicates stage 3 differentiated, preferentially neuronal-type differentiated
cells; adipo/osteo indicates cells
differentiated into adipocyte or osteoblast direction from MSC.
2) Occurrence of terminal epitopes in glycoconjugates and/or specifically in N-
glycans (N), 0-glycans (0),
and/or glycosphingolipids (L). Code: q, qualitative data; +/-, low expression;
+, common; ++, abundant.
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Table 22.
Examples of glycosphingolipid glycan classification
Class Definition
Lac nHeX - 2 1 1
Ltri nxex = 2 and nHexNAe = 1 18 25
Ll nxeX - 3 and nHexNAe 1 46 56
L2 3< nHex < 4 and nHexNAe = 2 11 <1
L3+ i+ l< nHe% < i+2 and nHexNAe i> 3 1 1
Gb nHex = 4 and nHexNAe = 1 20 16
0 other types 23 1
F fucosylated, ndHeX > 1 43 1
T non-reducing terminal HexNAc, 27 26
nHex C nHexNAc + 1
SAl monosialylated, nNe.5Ae 1 86
SA2 disialylated, nNe~Ae 2 14
SP sul hated or hos ho lated, +80 Da <1
Examples of 0-linked glycan classification
U U =~
Class Definition
W a~ W
01 nxex 1 and nHexNAe 1 E5 43
02 nHex 2 and nHexNAe 2 35
03+ nxex - i and nxexNAe i> 3 13
0 other types 9
F fucosylated, ndHeX > 1 1 64
T non-reducing terminal HexNAc, 12 <1
nHex C nHexNAc + 1
SAl monosialylated, nNe1sAo - 1 39
SA2 disialylated, nNe,5Ae = 2 52
SP sulphated or phosphorylated, +80 Da 8
a) not included in present quantitative analysis.
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Table 23.
Neutral
glycosphingolipid hESC
glycans#
L1 1
L2 64
L3 12
L4 1
L5+ 0.5
Gb 20
O 2
fucosylated 43
a1,2-Fuc 39
al,3/4-Fuc 3
(31,4-Gal 4
(31,3-Ga1 50
term. HexNAc 27
Acidic
glycosphingolipid hESC
glycans#
Ll n.d.
L2 81
L3 0.5
L4 0.5
L5+ 0.5
Gb 16
O <0.5
a-NeuAc 100
a2,3-NeuAc 81
fucosylated 1
11,4-Gal n.d.
#Abbreviations: Ll-6, glycosphingolipid glycan type Li, wherein nHexNAo + 1<
nHex < nHexNAo + 2, and i
nHeXNA~ + 1; Gb, (iso)globopentaose, wherein nxeX - 4 and nHexNA, - 1; term.
HexNAc, terminal HexNAc in Ll-
6, wherein nHe,u.rA, + 1= nHex; 0, other types; n.d., not determined.
Figures indicate percentage of total detected glycan signals.
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Table 24. One way ANOVA of acidic glycans from hESC, embryoid bodies and stage
3
stem cells. "x" denotes p-value < 0.05 and "y" equals 0.051 < p-value < 0.099.
P-values
highlighted with green or light green depict statistically significant down
regulation of
corresponding mass intensity. Due to low n number p-values < 0.099 were
considered to be
significant.
2068 x 2457
2074 2482 x
Mass hESC-EB hESC-st3 2076 y 2483
1354 x 2082 2512
1362 2092 2513
1403 2117 2521
1475 x 2133 x 2522
1500 x 2156 x 2528
1516 2157 2529
1541 x 2164 2544
1549 2174 2570
1557 2178 2571 y
1563 2214 2586
1565 2219 2587
1637 x 2221 2603
1678 x x 2222 2644 x x
1703 x x 2230 x x 2645 y
1709 2237 2660
1711 2238 2683 y
1717 2239 x 2714
1719 x y 2246 2732 y
1727 2253 y 2733
1744 y 2254 2791
1760 2263 y 2806
1768 y 2279 x x 2807 y
1791 x 2280 x 2812
1799 x 2293 2878
1840 2295 2879
1849 2302 2880
1856 2305 2886 x
1865 x 2319 2936
1873 x y 2320 2952
1889 y 2321 2953
1906 x x 2349 3024
1914 x 2365 3025
1928 E
67 ~~~~ ~\~`~\\\\\\\\\\\\\\\\\\\\\\\\~`~~~~~ ~~ 3026
1930 : 68 3098
1946 Y 0 76 30
99
1947 x x 2383 3104
1971 2384 y 3105
1972 2390 3170
2002 x y 2400 3171
2010 x 2406 3172
2011 2408 3244
2018 2424 3389
2035 x 2425 3390
2051 2441 y 3463
2052 2447 y y
2060 2448
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Table 25. One way ANOVA of N-glycans from hESC, embryoid bodies and stage 3
stem
cells. "x" denotes p-value < 0.05 and "y" equals 0.051 < p-value < 0.099. P-
values
highlighted with green or light green depict statistically significant down
regulation of
corresponding mass intensity. Due to low n number p-values < 0.099 were
considered to be
significant.
:.::.;::., ..: .... .... ...................... ..
1590
1~:::>::::>:'
.............................................................................
............................................................................
.............................................................................
............................................................................
.............................................................................
1606
::
609 1622
.............................................................
730 x 1647
771 x x 1663
892 x x 1688 x
917 x x 1702 x x
933 x y 1704
1031 1717
1054 x x 1743
1079 x x 1752
1095 x 1768
1120 y x 1784
1136 1793
1209 1809
1216 x y 1825 x
1241 1850 x
1257 y 1866
1282 y 1905
1298 1955
1323 1971
1339 y 1987
1378 x 1996 y y
1393 2012
1403 y 2028 \\\\\~\\\\\\\\\\\\\\\~\\\\\\
1419 2041
.... .... .... .... .... .... .... .... .... .... .... .... .
1428 2067
1444 2101
1460 2117
1485 2142
1501 y 2158 y
1540 x 2174
1555 2229
1565 y 2304 x
1581
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Table 26. Factor loadings for masses derived from acidic glycan of embryonic
stem cells.
Total of 13 factors were identified with Eigenvalues > 1 but 8 of them
explained approx > 5
% of all variation. Factors 1 to 8 explain 24.3%, 12.6%, 11%, 8.1%, 5.9%,
5.6%, 5.1%, and
4.7% of all variation, respectively.
Factor Factor Factor Factor Factor Factor Factor Factor
1 2 3 4 5 6 7 8
1354 0.10 -0.02 -0.03 -0.92 0.07 0.03 0.01 0.07
1362 0.10 0.11 -0.05 -0.01 0.11 -0.06 -0.48 0.02
1403 0.01 0.01 -0.02 -0.04 0.00 -0.07 0.07 0.01
1475 0.26 -0.88 -0.01 -0.09 -0.11 0.17 0.16 -0.10
1500 0.28 -0.37 0.10 -0.68 -0.25 0.27 0.01 -0.08
1516 0.31 0.11 -0.01 -0.78 0.05 -0.01 0.15 0.19
1541 -0.05 -0.14 -0.01 -0.92 -0.01 0.08 -0.21 0.03
1549 -0.06 0.15 0.19 0.12 0.11 0.07 0.06 0.04
1557 0.05 0.06 -0.06 -0.27 0.12 -0.03 0.09 0.00
1565 0.50 0.19 0.15 -0.23 0.36 -0.15 -0.03 0.40
1637 0.29 -0.80 0.02 -0.15 -0.08 0.20 0.08 -0.13
1678 0.79 -0.50 0.11 -0.14 0.02 0.04 0.08 0.01
1703 0.29 -0.28 0.03 -0.43 -0.44 0.13 0.07 0.17
1711 0.02 -0.20 -0.22 0.53 -0.02 0.35 -0.02 0.10
1719 0.30 0.19 0.05 -0.59 -0.45 0.10 0.10 0.07
1727 0.68 -0.28 0.11 0.07 0.55 -0.17 0.09 -0.12
1744 0.33 -0.25 0.04 -0.51 -0.06 0.09 -0.15 0.25
1768 0.51 0.25 0.00 -0.19 0.05 -0.10 -0.17 0.36
1791 -0.04 -0.12 -0.01 -0.98 0.00 0.07 0.09 0.01
1799 0.16 -0.90 -0.03 -0.02 0.12 0.10 -0.21 0.20
1840 0.57 -0.40 0.05 0.24 0.21 0.16 -0.08 0.40
1865 0.20 -0.17 0.01 -0.70 -0.07 0.06 0.02 0.02
1873 0.85 -0.25 0.12 -0.04 -0.04 0.29 -0.01 -0.05
1889 0.85 -0.06 0.18 -0.09 -0.03 0.00 0.18 0.05
1906 0.56 -0.43 0.07 -0.42 -0.02 0.06 -0.27 -0.15
1914 0.74 -0.14 0.17 -0.16 -0.07 0.34 -0.12 -0.19
1930 -0.15 0.55 0.06 0.23 0.30 -0.28 0.03 0.25
1946 0.04 0.27 0.20 0.20 0.00 -0.40 0.01 0.15
1947 0.44 -0.34 0.03 -0.38 -0.06 0.16 -0.09 -0.28
2002 0.77 -0.30 0.08 0.00 -0.06 0.23 0.09 -0.05
2010 0.21 -0.14 -0.03 -0.77 0.11 0.07 -0.03 0.09
2011 0.12 0.00 0.20 0.07 -0.73 -0.10 -0.13 0.05
2018 0.37 0.31 -0.05 0.07 0.22 -0.17 0.47 0.11
2035 0.56 -0.41 0.00 -0.19 0.22 0.09 0.09 0.38
2052 0.62 -0.31 0.16 -0.03 -0.09 0.33 0.01 -0.10
2068 0.35 -0.53 0.01 -0.60 0.13 0.12 -0.13 0.28
2076 -0.31 0.62 0.04 0.44 0.29 -0.04 -0.14 0.14
2092 -0.08 0.52 0.47 0.44 -0.04 -0.24 -0.09 -0.06
2117 0.25 -0.08 0.07 0.52 -0.12 0.31 -0.04 -0.33
2133 0.39 -0.69 -0.06 -0.23 0.33 -0.23 -0.05 -0.11
2156 0.33 -0.14 0.04 -0.79 0.04 0.04 0.06 -0.03
2157 -0.15 -0.05 0.38 0.17 0.03 -0.07 0.30 0.32
2164 0.22 0.22 0.13 -0.14 -0.12 -0.49 -0.53 0.29
2221 -0.19 0.21 -0.86 0.19 0.12 -0.16 0.09 0.06
2222 -0.52 0.27 0.63 0.33 0.03 0.02 0.09 0.04
2230 0.25 -0.10 0.07 -0.65 -0.43 0.19 -0.14 0.08
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2237 0.12 0.30 0.12 0.22 0.18 -0.40 0.04 -0.34
2238 -0.23 -0.06 0.63 0.09 -0.34 0.56 0.16 0.10
2239 0.18 0.03 0.06 0.12 -0.31 0.16 -0.44 -0.35
2246 -0.01 -0.01 -0.03 -0.72 0.09 0.04 0.44 -0.09
2253 -0.01 0.20 0.07 0.09 0.03 -0.38 0.03 0.03
2254 -0.20 0.01 0.07 0.05 -0.11 -0.91 -0.02 0.00
2263 -0.12 -0.14 0.53 0.39 -0.11 0.11 0.11 -0.20
2279 0.12 -0.35 -0.77 0.03 0.11 0.22 -0.16 -0.15
2280 0.22 -0.44 -0.65 0.07 0.34 -0.04 0.11 0.10
2295 0.29 0.42 0.23 0.02 0.20 -0.18 -0.52 -0.31
2321 0.07 -0.02 0.13 0.02 -0.86 -0.30 0.00 -0.09
2367 -0.65 0.44 -0.21 0.44 0.17 -0.02 0.14 0.10
2368 -0.31 0.27 0.57 0.20 0.32 -0.33 0.18 -0.23
2383 -0.01 0.19 0.18 0.18 -0.02 -0.67 -0.15 0.15
2384 0.10 0.22 0.17 0.16 -0.49 -0.08 -0.01 0.06
2390 -0.31 0.23 0.41 0.10 0.12 -0.30 -0.09 0.17
2400 0.11 -0.02 0.04 0.21 -0.36 0.10 0.08 -0.85
2408 -0.52 0.19 0.54 0.32 -0.22 0.00 0.12 0.13
2425 0.09 -0.39 0.54 0.20 -0.24 0.22 0.12 -0.29
2441 -0.77 0.15 -0.09 0.48 0.05 0.19 -0.05 -0.06
2447 0.30 0.23 0.03 -0.68 0.10 0.07 -0.20 0.19
2448 0.26 0.15 -0.04 -0.30 0.12 -0.02 -0.09 0.16
2482 0.34 -0.74 0.03 -0.18 -0.25 0.22 0.10 -0.12
2512 0.07 0.07 -0.04 -0.03 0.06 -0.08 -0.25 0.02
2513 0.10 0.12 -0.04 0.01 0.13 -0.03 -0.59 0.02
2521 0.30 -0.14 0.13 -0.35 -0.26 -0.12 0.00 0.26
2522 0.09 -0.01 -0.02 -0.19 -0.12 0.06 0.02 -0.01
2528 -0.15 0.05 0.05 0.05 -0.05 -0.88 -0.24 0.00
2529 0.34 0.18 0.02 0.09 -0.03 0.02 -0.10 0.25
2544 -0.20 0.01 0.07 0.04 -0.11 -0.91 -0.02 0.00
2570 0.00 0.06 -0.74 0.10 0.10 -0.12 -0.11 -0.12
2571 -0.14 0.08 -0.70 -0.18 -0.28 0.18 0.36 -0.35
2586 0.15 0.24 0.07 0.02 0.00 0.04 -0.16 0.08
2587 -0.55 0.15 0.67 0.21 0.01 0.02 0.13 -0.02
2603 0.02 -0.02 0.07 0.14 -0.90 0.13 0.20 -0.13
2644 -0.07 -0.33 -0.86 -0.06 -0.05 0.23 0.00 -0.05
2645 -0.22 -0.03 -0.90 0.16 0.07 0.10 0.05 0.01
2660 -0.07 0.14 0.20 0.13 0.11 0.09 0.04 0.03
2683 0.25 -0.37 0.04 -0.23 -0.36 0.21 -0.15 0.18
2714 0.14 -0.70 -0.08 0.26 0.23 -0.01 0.18 0.20
2732 -0.68 0.32 -0.53 0.09 0.12 0.01 0.04 0.24
2733 -0.02 0.06 0.36 0.27 0.53 0.25 0.31 -0.07
2807 -0.80 -0.04 -0.18 0.23 0.08 0.18 0.32 -0.24
2878 0.20 -0.04 0.02 0.23 0.22 0.13 0.25 0.14
2879 -0.03 0.04 0.02 0.09 0.07 -0.61 -0.15 -0.50
2880 -0.68 0.07 0.46 0.16 0.18 0.19 0.13 0.14
2886 0.13 -0.41 -0.01 -0.58 0.15 0.10 0.07 0.17
2936 -0.26 0.24 -0.87 0.16 0.05 0.04 0.16 0.13
2953 -0.59 0.12 0.44 0.21 0.09 -0.49 0.07 0.10
3024 0.19 0.21 -0.04 -0.48 0.19 -0.31 0.64 0.01
3025 0.09 0.21 0.02 0.10 0.07 -0.82 0.29 0.07
3098 -0.35 0.20 -0.86 0.17 0.01 0.14 0.05 0.10
3099 -0.74 0.09 0.48 0.12 0.11 -0.35 0.02 0.09
3170 0.12 -0.01 -0.01 0.14 0.19 0.02 -0.04 -0.90
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3171 0.01 0.01 -0.02 -0.04 0.00 -0.07 0.07 0.01
3172 -0.72 0.07 0.47 0.18 0.13 -0.16 0.11 0.13
3390 -0.01 0.15 0.05 0.09 0.01 -0.92 0.18 0.05
3463 -0.08 0.20 0.13 0.15 0.01 -0.29 0.00 0.01
Expl.Var 13.78 9.49 10.82 12.50 5.86 8.57 3.89 4.66
Prp.Totl 0.13 0.09 0.10 0.12 0.06 0.08 0.04 0.04
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Table 27. Communalities for masses derived from acidic glycan of embryonic
stem cells.
COMMUNALITIES
From 1 From 2 From 3 From 4 From 5 From 6 From 7 From 8
Factor Factors Factors Factors Factors Factors Factors Factors
1354 0.009 0.009 0.010 0.860 0.865 0.866 0.866 0.870
1362 0.010 0.022 0.024 0.024 0.037 0.041 0.276 0.276
1403 0.000 0.000 0.001 0.003 0.003 0.008 0.012 0.012
1475 0.067 0.845 0.845 0.854 0.866 0.895 0.920 0.931
1500 0.076 0.216 0.226 0.692 0.753 0.826 0.827 0.833
1516 0.093 0.105 0.105 0.708 0.710 0.710 0.732 0.769
1541 0.002 0.022 0.022 0.876 0.876 0.882 0.927 0.928
1549 0.004 0.025 0.062 0.076 0.088 0.093 0.096 0.097
1557 0.003 0.007 0.010 0.081 0.095 0.096 0.104 0.104
1565 0.249 0.284 0.308 0.360 0.488 0.510 0.510 0.674
1637 0.086 0.732 0.732 0.755 0.761 0.801 0.807 0.823
1678 0.626 0.871 0.883 0.902 0.902 0.904 0.911 0.911
1703 0.085 0.163 0.164 0.351 0.548 0.564 0.569 0.599
1711 0.000 0.039 0.088 0.373 0.374 0.495 0.495 0.505
1719 0.088 0.126 0.128 0.482 0.684 0.694 0.704 0.708
1727 0.469 0.545 0.556 0.562 0.860 0.890 0.898 0.914
1744 0.108 0.170 0.172 0.437 0.440 0.448 0.470 0.530
1768 0.263 0.327 0.327 0.363 0.365 0.374 0.404 0.533
1791 0.001 0.016 0.016 0.968 0.968 0.973 0.982 0.982
1799 0.024 0.832 0.833 0.834 0.849 0.859 0.903 0.942
1840 0.326 0.486 0.489 0.546 0.591 0.618 0.625 0.785
1865 0.042 0.071 0.071 0.564 0.569 0.572 0.572 0.573
1873 0.714 0.776 0.791 0.793 0.795 0.880 0.880 0.882
1889 0.726 0.730 0.761 0.769 0.770 0.770 0.803 0.806
1906 0.319 0.507 0.513 0.690 0.690 0.694 0.766 0.787
1914 0.549 0.568 0.596 0.621 0.625 0.742 0.758 0.795
1930 0.022 0.326 0.330 0.384 0.471 0.552 0.553 0.616
1946 0.001 0.075 0.114 0.154 0.154 0.315 0.315 0.338
1947 0.193 0.312 0.313 0.455 0.459 0.484 0.492 0.569
2002 0.591 0.682 0.688 0.688 0.692 0.745 0.753 0.755
2010 0.045 0.065 0.066 0.666 0.678 0.683 0.685 0.694
2011 0.015 0.015 0.054 0.059 0.595 0.605 0.621 0.623
2018 0.136 0.231 0.234 0.240 0.286 0.313 0.530 0.542
2035 0.313 0.477 0.477 0.514 0.560 0.568 0.577 0.720
2052 0.378 0.471 0.498 0.498 0.507 0.615 0.615 0.625
2068 0.123 0.402 0.402 0.758 0.775 0.788 0.807 0.886
2076 0.097 0.485 0.487 0.677 0.760 0.761 0.782 0.801
2092 0.007 0.282 0.506 0.701 0.702 0.759 0.767 0.771
2117 0.064 0.069 0.074 0.343 0.357 0.455 0.457 0.568
2133 0.156 0.631 0.634 0.689 0.798 0.849 0.852 0.865
2156 0.108 0.129 0.131 0.755 0.757 0.759 0.762 0.763
2157 0.021 0.024 0.171 0.201 0.202 0.207 0.296 0.401
2164 0.048 0.096 0.113 0.134 0.148 0.387 0.669 0.754
2221 0.038 0.083 0.821 0.858 0.874 0.898 0.906 0.909
2222 0.269 0.343 0.735 0.847 0.848 0.848 0.856 0.858
2230 0.063 0.073 0.078 0.497 0.684 0.720 0.741 0.747
2237 0.014 0.103 0.117 0.166 0.197 0.360 0.361 0.477
2238 0.054 0.057 0.451 0.460 0.578 0.893 0.920 0.931
2239 0.033 0.034 0.038 0.052 0.145 0.171 0.365 0.485
2246 0.000 0.000 0.001 0.515 0.524 0.525 0.721 0.729
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2253 0.000 0.041 0.047 0.055 0.056 0.201 0.202 0.203
2254 0.040 0.040 0.045 0.047 0.058 0.893 0.893 0.893
2263 0.015 0.033 0.312 0.461 0.473 0.486 0.498 0.537
2279 0.014 0.134 0.733 0.734 0.746 0.795 0.822 0.845
2280 0.047 0.240 0.667 0.671 0.788 0.790 0.801 0.811
2295 0.082 0.254 0.307 0.308 0.347 0.379 0.647 0.742
2321 0.004 0.005 0.022 0.022 0.761 0.851 0.851 0.859
2367 0.421 0.612 0.658 0.855 0.885 0.886 0.906 0.915
2368 0.094 0.166 0.487 0.526 0.630 0.742 0.774 0.827
2383 0.000 0.037 0.071 0.103 0.103 0.548 0.569 0.591
2384 0.010 0.058 0.086 0.112 0.353 0.359 0.359 0.362
2390 0.097 0.149 0.315 0.324 0.337 0.428 0.436 0.463
2400 0.012 0.012 0.013 0.056 0.184 0.194 0.200 0.919
2408 0.275 0.311 0.603 0.705 0.755 0.755 0.769 0.787
2425 0.008 0.158 0.447 0.487 0.544 0.592 0.606 0.689
2441 0.591 0.614 0.623 0.857 0.859 0.894 0.897 0.900
2447 0.093 0.148 0.149 0.618 0.627 0.632 0.672 0.706
2448 0.068 0.091 0.093 0.182 0.195 0.196 0.205 0.229
2482 0.113 0.654 0.655 0.688 0.750 0.798 0.807 0.821
2512 0.004 0.010 0.011 0.012 0.016 0.022 0.082 0.083
2513 0.011 0.024 0.026 0.026 0.043 0.043 0.390 0.391
2521 0.091 0.110 0.125 0.245 0.311 0.327 0.327 0.393
2522 0.008 0.008 0.009 0.047 0.062 0.065 0.066 0.066
2528 0.023 0.026 0.029 0.031 0.034 0.814 0.872 0.872
2529 0.117 0.151 0.151 0.160 0.160 0.161 0.171 0.233
2544 0.039 0.039 0.044 0.046 0.057 0.883 0.883 0.883
2570 0.000 0.004 0.557 0.566 0.577 0.590 0.603 0.618
2571 0.019 0.026 0.510 0.541 0.618 0.650 0.777 0.901
2586 0.022 0.078 0.083 0.083 0.083 0.085 0.111 0.118
2587 0.298 0.320 0.774 0.818 0.818 0.818 0.835 0.836
2603 0.000 0.001 0.006 0.027 0.838 0.854 0.896 0.915
2644 0.005 0.111 0.845 0.849 0.851 0.904 0.904 0.906
2645 0.049 0.050 0.867 0.892 0.897 0.908 0.910 0.910
2660 0.005 0.025 0.065 0.083 0.096 0.103 0.105 0.106
2683 0.062 0.198 0.199 0.250 0.380 0.424 0.447 0.481
2714 0.020 0.513 0.519 0.586 0.639 0.639 0.671 0.710
2732 0.460 0.563 0.839 0.848 0.863 0.863 0.865 0.922
2733 0.000 0.004 0.135 0.207 0.489 0.552 0.646 0.651
2807 0.632 0.634 0.666 0.720 0.727 0.760 0.864 0.920
2878 0.041 0.043 0.043 0.094 0.143 0.160 0.223 0.241
2879 0.001 0.002 0.003 0.011 0.016 0.384 0.407 0.659
2880 0.457 0.463 0.674 0.701 0.734 0.770 0.788 0.807
2886 0.017 0.189 0.189 0.522 0.543 0.553 0.558 0.586
2936 0.067 0.123 0.885 0.911 0.913 0.915 0.942 0.957
2953 0.348 0.363 0.557 0.602 0.611 0.852 0.856 0.866
3024 0.037 0.079 0.081 0.314 0.350 0.448 0.862 0.862
3025 0.008 0.055 0.055 0.065 0.069 0.748 0.830 0.835
3098 0.123 0.165 0.897 0.927 0.928 0.946 0.948 0.959
3099 0.552 0.560 0.791 0.806 0.819 0.945 0.945 0.954
3170 0.013 0.013 0.013 0.033 0.071 0.072 0.073 0.888
3171 0.000 0.000 0.001 0.003 0.003 0.008 0.012 0.012
3172 0.523 0.527 0.747 0.779 0.796 0.823 0.835 0.851
3390 0.000 0.023 0.025 0.034 0.034 0.878 0.911 0.913
3463 0.006 0.044 0.060 0.081 0.081 0.168 0.168 0.168
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Table 28. Factor loadings for masses derived from neutral N-glycan of
embryonic stem
cells. Factors representing Eigenvalues > 1 are shown. Factors 1 to 7 explain
26.30%,
15.30%, 11.04%, 10.09%, 7.59%, 7.27% and 4.45% of all variation, respectively.
hESC Varimax normalised
% 26.30 15.30 11.04 10.09 7.59 7.27 4.45
explained
Factor Factor Factor 3 Factor 4 Factor 5 Factor 6 Factor 7
1 2
609 -0.79 0.00 0.14 0.03 -0.30 0.11 -0.10
730 0.28 0.06 -0.21 0.40 0.77 -0.10 0.00
771 0.72 0.07 -0.34 0.05 0.47 0.09 -0.05
892 0.81 -0.05 -0.14 0.20 0.42 -0.11 0.08
917 0.46 0.02 -0.62 -0.19 0.46 0.34 -0.02
933 0.81 0.01 -0.34 -0.15 0.01 0.31 0.20
1031 0.13 0.02 -0.03 -0.04 0.69 -0.06 0.07
1054 0.78 -0.03 -0.21 0.06 0.21 -0.05 0.04
0.51 -0.21 -0.60 -0.20 0.35 0.37 0.12
1095 0.78 0.03 -0.13 0.13 -0.08 0.46 0.21
1120 0.37 0.16 -0.88 0.16 0.05 0.02 -0.14
1136 0.14 -0.16 0.11 0.82 -0.07 -0.41 0.03
1209 0.06 -0.01 -0.05 0.89 0.03 -0.18 0.06
1216 0.86 0.20 0.08 0.26 0.03 0.15 0.04
1241 0.24 0.12 -0.71 0.09 -0.05 0.56 0.18
1257 0.11 -0.52 0.08 -0.25 0.13 0.69 -0.25
1282 0.13 -0.14 -0.91 0.18 0.07 -0.09 0.05
1298 0.09 -0.38 0.78 0.10 -0.23 0.11 0.09
1339 0.25 0.10 -0.81 -0.27 0.17 -0.12 -0.17
1378 0.86 0.22 -0.12 0.10 -0.30 0.13 -0.07
-0.46 0.05 0.17 0.24 -0.05 0.58 -0.04
1403 0.31 -0.09 -0.81 -0.16 0.12 0.34 -0.02
-0.30 0.43 0.09 -0.47 -0.11 0.56 0.19
1444 -0.14 0.03 -0.61 0.01 -0.54 0.17 -0.23
1460 0.12 -0.77 0.51 -0.11 -0.22 -0.13 -0.15
1485 -0.17 -0.80 0.27 0.06 0.32 -0.02 0.14
1501 0.32 0.10 -0.82 -0.23 0.25 -0.19 -0.12
1540 0.82 0.17 0.23 0.22 -0.24 -0.06 -0.31
-0.08 0.23 0.18 0.00 0.33 0.28 0.38
1565 0.11 -0.12 -0.20 0.10 0.79 0.42 -0.14
1581 -0.66 0.58 0.03 -0.20 0.09 0.21 0.09
1590 0.09 0.33 0.12 0.67 0.12 0.27 0.08
1606 -0.15 -0.81 0.10 0.08 -0.01 0.25 0.44
1622 0.13 -0.79 0.42 -0.10 -0.23 -0.08 -0.25
1647 -0.45 -0.67 0.18 0.22 0.02 0.38 0.30
: _ .. -0.50 -0.42 0.23 0.17 -0.52 -0.26 -0.06
1688 -0.18 -0.38 -0.19 0.24 0.64 0.31 0.01
1702 0.85 0.09 0.05 0.35 -0.17 0.02 -0.18
1704 0.00 -0.88 -0.02 -0.18 -0.21 0.00 0.32
0.12 0.16 0.17 0.39 0.36 0.11 0.37
1743 -0.74 0.37 0.32 -0.08 0.08 -0.39 -0.12
1 2 0.08 0.09 0.03 -0.05 0.02 0.31 0.02
0;.= -0.05 -0.48 -0.16 0.11 0.27 -0.20 -0.15
1784 0.24 -0.18 0.15 -0.10 0.03 -0.13 0.65
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1793 0.30 0.36 -0.72 0.03 -0.13 -0.14 -0.17
1809 -0.78 -0.11 0.14 -0.05 -0.48 0.12 -0.05
1825 0.03 -0.21 -0.41 -0.23 0.67 -0.37 -0.22
1850 0.02 -0.90 -0.19 0.17 0.24 0.09 -0.03
1866 0.11 -0.86 0.04 -0.31 -0.06 -0.22 0.11
1905 -0.28 0.25 0.32 0.01 -0.26 -0.80 0.06
1955 -0.83 0.32 0.17 0.20 -0.07 -0.02 -0.17
-0.06 -0.52 -0.01 0.47 0.03 0.30 -0.07
1987 0.24 0.16 -0.67 0.03 -0.25 -0.46 -0.19
1996 0.14 0.07 -0.86 -0.06 0.19 0.05 -0.32
2012 0.14 -0.71 0.16 0.30 -0.24 0.21 0.47
2028 -0.73 0.11 0.35 0.07 -0.32 0.10 0.33
-0.32 -0.08 0.33 0.45 -0.20 0.02 0.68
-0.05 0.29 0.55 -0.22 -0.32 -0.52 0.32
-0.37 0.47 -0.40 0.10 -0.41 0.35 -0.06
2117 0.03 0.05 -0.02 -0.09 0.63 0.04 -0.06
2 0.31 0.17 0.63 -0.15 -0.21 -0.07 -0.46
2158 0.20 -0.04 0.05 0.82 0.13 0.31 0.04
2174 -0.79 0.16 0.32 0.04 -0.32 0.12 0.24
2229 -0.04 -0.37 0.22 0.24 -0.19 -0.07 0.79
2304 0.12 -0.03 -0.21 0.21 0.85 0.24 -0.10
2:._.. -0.57 0.42 0.11 0.46 -0.12 0.00 0.08
2391 0.03 -0.06 0.00 0.86 0.13 0.10 0.10
2393 0.26 0.12 0.00 0.14 0.33 0.02 0.25
2466 0.05 -0.07 -0.07 0.85 -0.05 -0.27 0.02
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Table 29. Communalities for masses derived from neutral N-glycans of embryonic
stem
cells.
From 1 From 2 From 3 From 4 From 5 From 6 From 7
Factor Factors Factors Factors Factors Factors Factors
609 0.618 0.618 0.639 0.640 0.733 0.745 0.755
730 0.080 0.084 0.128 0.286 0.876 0.887 0.887
771 0.525 0.531 0.648 0.650 0.874 0.883 0.885
892 0.663 0.665 0.684 0.724 0.901 0.914 0.921
917 0.209 0.209 0.591 0.626 0.838 0.953 0.953
933 0.649 0.650 0.765 0.789 0.789 0.885 0.925
1031 0.016 0.016 0.017 0.019 0.492 0.496 0.500
1054 0.605 0.606 0.648 0.651 0.696 0.698 0.699
1079 0.257 0.301 0.663 0.701 0.824 0.959 0.973
1095 0.609 0.610 0.628 0.644 0.651 0.865 0.908
1120 0.136 0.162 0.936 0.962 0.965 0.965 0.985
1136 0.020 0.045 0.057 0.722 0.727 0.896 0.897
1209 0.003 0.003 0.006 0.791 0.792 0.823 0.827
1216 0.741 0.780 0.786 0.853 0.854 0.875 0.877
1241 0.058 0.072 0.577 0.586 0.589 0.897 0.929
1257 0.012 0.282 0.288 0.349 0.365 0.847 0.912
1282 0.017 0.037 0.862 0.895 0.901 0.909 0.911
1298 0.009 0.156 0.763 0.773 0.825 0.838 0.845
1339 0.063 0.073 0.731 0.802 0.830 0.843 0.873
1378 0.736 0.783 0.797 0.808 0.901 0.919 0.924
1393 0.213 0.215 0.244 0.301 0.304 0.641 0.642
1403 0.093 0.101 0.764 0.789 0.804 0.918 0.918
1419 0.093 0.280 0.288 0.510 0.522 0.831 0.868
1444 0.020 0.021 0.394 0.394 0.683 0.712 0.764
1460 0.014 0.608 0.867 0.879 0.927 0.945 0.966
1485 0.029 0.661 0.732 0.736 0.835 0.835 0.854
1501 0.104 0.113 0.780 0.835 0.895 0.930 0.944
1540 0.667 0.695 0.747 0.795 0.854 0.857 0.952
1555 0.007 0.059 0.093 0.093 0.203 0.283 0.424
1565 0.013 0.028 0.067 0.076 0.695 0.874 0.894
1581 0.430 0.767 0.768 0.810 0.818 0.861 0.868
1590 0.007 0.118 0.132 0.583 0.597 0.672 0.679
1606 0.022 0.672 0.682 0.688 0.688 0.753 0.944
1622 0.016 0.638 0.810 0.820 0.871 0.878 0.939
1647 0.198 0.647 0.678 0.728 0.728 0.871 0.961
1663 0.253 0.425 0.478 0.508 0.780 0.849 0.852
1688 0.034 0.176 0.210 0.266 0.679 0.773 0.773
1702 0.730 0.738 0.740 0.865 0.895 0.896 0.927
1704 0.000 0.768 0.768 0.802 0.847 0.847 0.951
1717 0.015 0.040 0.071 0.219 0.350 0.363 0.499
1743 0.554 0.689 0.789 0.796 0.802 0.957 0.970
1752 0.007 0.015 0.016 0.018 0.019 0.112 0.112
1768 0.003 0.229 0.254 0.268 0.339 0.380 0.401
1784 0.057 0.089 0.111 0.122 0.123 0.141 0.559
1793 0.088 0.215 0.729 0.730 0.748 0.768 0.797
1809 0.604 0.616 0.635 0.638 0.867 0.883 0.885
1825 0.001 0.045 0.212 0.266 0.714 0.852 0.901
1850 0.000 0.803 0.838 0.866 0.925 0.934 0.935
1866 0.012 0.748 0.750 0.847 0.850 0.898 0.911
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1905 0.077 0.139 0.244 0.244 0.310 0.952 0.955
1955 0.683 0.787 0.816 0.857 0.862 0.863 0.890
1971 0.004 0.272 0.273 0.491 0.492 0.580 0.584
1987 0.059 0.084 0.536 0.537 0.598 0.806 0.842
1996 0.020 0.026 0.768 0.772 0.809 0.812 0.914
2012 0.021 0.524 0.549 0.641 0.698 0.743 0.962
2028 0.531 0.543 0.664 0.669 0.772 0.782 0.887
2041 0.104 0.111 0.221 0.427 0.469 0.469 0.935
2067 0.002 0.088 0.393 0.440 0.544 0.812 0.914
2101 0.140 0.362 0.521 0.531 0.700 0.822 0.826
2117 0.001 0.003 0.004 0.011 0.409 0.411 0.415
2142 0.095 0.125 0.519 0.543 0.586 0.592 0.799
2158 0.040 0.041 0.043 0.715 0.732 0.827 0.829
2174 0.627 0.654 0.757 0.759 0.859 0.874 0.934
2229 0.001 0.135 0.181 0.240 0.277 0.282 0.913
2304 0.015 0.016 0.061 0.107 0.822 0.878 0.889
2320 0.329 0.502 0.513 0.720 0.734 0.734 0.741
2391 0.001 0.004 0.004 0.744 0.760 0.771 0.781
2393 0.069 0.082 0.082 0.102 0.211 0.212 0.275
2466 0.003 0.008 0.013 0.744 0.746 0.817 0.818
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Table 30. Correlation matrix for neutral glycans derived from embryonic stem
cells.
730 771 892 917 933 1054 1079 1095 1120 1136 1216 1241 1257 1282 1298 1323
1339 1378
730 1.00 0.69 0.68 0.53 0.22 0.54 0.41 0.14 0.40 0.27 0.35 0.15 -0.09 0.33 -
0.28 0.17 0.30 0.08
771 0.69 1.00 0.83 0.84 0.78 0.74 0.77 0.61 0.63 0.04 0.56 0.47 0.09 0.39 -
0.22 0.40 0.53 0.53
892 0.68 0.83 1.00 0.58 0.64 0.84 0.60 0.58 0.46 0.29 0.75 0.24 -0.01 0.34 -
0.08 0.20 0.37 0.58
917 0.53 0.84 0.58 1.00 0.74 0.59 0.95 0.50 0.72 -0.31 0.33 0.70 0.30 0.56 -
0.47 0.57 0.70 0.36
933 0.22 0.78 0.64 0.74 1.00 0.62 0.79 0.87 0.54 -0.15 0.63 0.61 0.23 0.29 -
0.10 0.33 0.46 0.73
1054 0.54 0.74 0.84 0.59 0.62 1.00 0.61 0.50 0.48 0.18 0.70 0.35 0.08 0.38 -
0.08 0.33 0.38 0.59
1079 0.41 0.77 0.60 0.95 0.79 0.61 1.00 0.59 0.65 -0.27 0.35 0.72 0.42 0.60 -
0.35 0.48 0.64 0.37
1095 0.14 0.61 0.58 0.50 0.87 0.50 0.59 1.00 0.41 0.06 0.73 0.61 0.32 0.19
0.07 0.14 0.18 0.74
1120 0.40 0.63 0.46 0.72 0.54 0.48 0.65 0.41 1.00 0.04 0.31 0.74 -0.10 0.87 -
0.71 0.82 0.83 0.46
1136 0.27 0.04 0.29 -0.31 -0.15 0.18 -0.27 0.06 0.04 1.00 0.20 -0.16 -0.38
0.11 0.28 -0.12 -0.27 0.08
1216 0.35 0.56 0.75 0.33 0.63 0.70 0.35 0.73 0.31 0.20 1.00 0.26 0.03 0.08
0.01 0.07 0.04 0.88
1241 0.15 0.47 0.24 0.70 0.61 0.35 0.72 0.61 0.74 -0.16 0.26 1.00 0.22 0.68 -
0.44 0.52 0.48 0.38
1257 -0.09 0.09 -0.01 0.30 0.23 0.08 0.42 0.32 -0.10 -0.38 0.03 0.22 1.00 -
0.09 0.25 -0.11 0.01 -0.03
1282 0.33 0.39 0.34 0.56 0.29 0.38 0.60 0.19 0.87 0.11 0.08 0.68 -0.09 1.00 -
0.69 0.71 0.71 0.17
1298 -0.28 -0.22 -0.08 -0.47 -0.10 -0.08 -0.35 0.07 -0.71 0.28 0.01 -0.44 0.25
-0.69 1.00 -0.70 -0.73 -0.05
1323 0.17 0.40 0.20 0.57 0.33 0.33 0.48 0.14 0.82 -0.12 0.07 0.52 -0.11 0.71 -
0.70 1.00 0.76 0.29
1339 0.30 0.53 0.37 0.70 0.46 0.38 0.64 0.18 0.83 -0.27 0.04 0.48 0.01 0.71 -
0.73 0.76 1.00 0.20
1378 0.08 0.53 0.58 0.36 0.73 0.59 0.37 0.74 0.46 0.08 0.88 0.38 -0.03 0.17 -
0.05 0.29 0.20 1.00
1393 -0.10 -0.36 -0.43 -0.22 -0.26 -0.57 -0.24 -0.10 -0.25 -0.25 -0.16 -0.01
0.24 -0.29 0.08 -0.48 -0.31 -0.20
1403 0.20 0.58 0.29 0.82 0.65 0.32 0.84 0.52 0.82 -0.29 0.15 0.84 0.32 0.76 -
0.57 0.73 0.73 0.32
1419 -0.50 -0.31 -0.52 -0.01 -0.02 -0.42 -0.05 0.08 -0.22 -0.67 -0.21 0.25
0.22 -0.28 -0.12 -0.08 -0.14 -0.15
1444 -0.35 -0.07 -0.24 0.14 0.09 -0.05 0.17 0.04 0.50 -0.06 -0.16 0.58 -0.03
0.51 -0.32 0.54 0.31 0.16
1460 -0.29 -0.22 -0.02 -0.38 -0.12 -0.05 -0.19 -0.10 -0.53 0.16 -0.09 -0.54
0.37 -0.39 0.75 -0.47 -0.40 -0.08
1485 0.05 -0.18 0.08 -0.16 -0.29 -0.05 0.04 -0.21 -0.42 0.18 -0.18 -0.28 0.37 -
0.03 0.39 -0.41 -0.36 -0.42
1501 0.41 0.63 0.45 0.75 0.53 0.44 0.69 0.20 0.83 -0.21 0.13 0.47 -0.07 0.72 -
0.75 0.79 0.95 0.26
1540 0.09 0.40 0.57 0.06 0.45 0.53 0.06 0.54 0.19 0.29 0.84 -0.02 -0.05 -0.09
0.17 0.05 -0.02 0.86
1555 0.16 0.06 0.04 0.08 0.13 -0.22 0.09 0.08 -0.24 -0.22 0.12 0.00 -0.04 -
0.28 -0.02 -0.39 -0.23 -0.06
1565 0.66 0.53 0.41 0.65 0.20 0.32 0.59 0.18 0.28 -0.17 0.24 0.35 0.43 0.30 -
0.29 0.14 0.17 -0.01
1 581 -0.26 -0.40 -0.61 -0.19 -0.44 -0.60 -0.34 -0.36 -0.19 -0.41 -0.51 0.06 -
0.17 -0.18 -0.28 -0.06 -0.11 -0.51
1590 0.43 0.07 0.19 -0.02 -0.02 0.21 -0.08 0.18 0.07 0.33 0.45 0.14 -0.06 -
0.03 -0.01 -0.21 -0.22 0.20
1606 -0.16 -0.24 -0.09 -0.12 -0.05 -0.11 0.15 0.10 -0.31 0.12 -0.20 0.07 0.48
0.07 0.41 -0.39 -0.34 -0.32
1622 -0.27 -0.18 -0.05 -0.31 -0.07 -0.08 -0.14 -0.07 -0.44 0.11 -0.08 -0.49
0.42 -0.34 0.66 -0.38 -0.33 -0.02
1647 -0.13 -0.37 -0.34 -0.21 -0.28 -0.30 -0.04 -0.14 -0.43 0.13 -0.39 0.00
0.44 -0.10 0.48 -0.51 -0.51 -0.50
1663 -0.54 -0.76 -0.62 -0.72 -0.63 -0.46 -0.62 -0.50 -0.45 0.36 -0.55 -0.44 -
0.04 -0.21 0.42 -0.28 -0.42 -0.45
1688 0.46 0.18 0.24 0.36 -0.12 0.12 0.39 -0.02 0.14 0.05 -0.07 0.22 0.40 0.36 -
0.15 -0.04 0.10 -0.33
1702 0.22 0.54 0.69 0.24 0.57 0.65 0.26 0.65 0.35 0.38 0.89 0.20 -0.05 0.12
0.11 0.12 0.05 0.91
1704 -0.28 -0.14 -0.06 -0.05 0.12 -0.06 0.21 0.04 -0.22 0.01 -0.22 -0.04 0.34
0.08 0.41 -0.19 -0.15 -0.13
1717 0.29 0.16 0.18 0.02 0.12 -0.16 -0.01 0.37 -0.04 0.26 0.26 0.04 -0.10 -
0.08 0.01 -0.22 -0.24 0.06
1743 -0.19 -0.62 -0.62 -0.62 -0.85 -0.65 -0.78 -0.82 -0.48 -0.05 -0.65 -0.59 -
0.45 -0.39 -0.05 -0.22 -0.30 -0.69
1768 0.25 0.07 0.02 0.05 -0.05 0.01 0.12 -0.06 0.04 0.25 -0.19 -0.07 0.24 0.20
-0.08 0.15 0.08 -0.30
1793 0.21 0.44 0.21 0.51 0.48 0.25 0.39 0.25 0.79 -0.03 0.24 0.44 -0.30 0.53 -
0.64 0.77 0.69 0.49
1809 -0.60 -0.79 -0.84 -0.56 -0.62 -0.68 -0.54 -0.62 -0.45 -0.17 -0.67 -0.26 -
0.02 -0.30 0.23 -0.34 -0.38 -0.51
1825 0.54 0.37 0.38 0.44 0.01 0.21 0.40 -0.24 0.36 -0.17 -0.09 -0.07 0.04 0.46
-0.57 0.41 0.61 -0.21
1850 0.24 0.11 0.16 0.20 0.06 0.08 0.37 0.05 0.06 0.16 -0.06 0.04 0.51 0.33
0.10 -0.02 0.03 -0.15
1866 -0.16 -0.08 0.06 -0.05 0.08 0.00 0.17 -0.07 -0.22 -0.07 -0.13 -0.28 0.31
0.03 0.29 -0.11 -0.03 -0.08
1905 -0.24 -0.51 -0.31 -0.73 -0.55 -0.38 -0.78 -0.56 -0.39 0.29 -0.26 -0.73 -
0.74 -0.35 0.07 -0.16 -0.27 -0.23
1955 -0.14 -0.63 -0.73 -0.51 -0.76 -0.66 -0.67 -0.73 -0.37 -0.01 -0.58 -0.35 -
0.30 -0.34 -0.02 -0.25 -0.36 -0.57
1996 0.32 0.46 0.26 0.69 0.36 0.25 0.61 0.18 0.86 -0.20 0.12 0.57 0.07 0.77 -
0.83 0.82 0.83 0.25
2012 -0.09 -0.10 0.07 -0.15 0.19 0.08 0.12 0.31 -0.24 0.33 0.12 0.06 0.33 -
0.02 0.56 -0.44 -0.40 0.05
2028 -0.46 -0.79 -0.74 -0.69 -0.61 -0.63 -0.66 -0.43 -0.61 0.00 -0.54 -0.25 -
0.14 -0.45 0.30 -0.50 -0.59 -0.57
2041 -0.15 -0.47 -0.24 -0.57 -0.29 -0.33 -0.42 -0.05 -0.47 0.41 -0.13 -0.13 -
0.25 -0.24 0.40 -0.64 -0.62 -0.30
2067 -0.46 -0.46 -0.26 -0.68 -0.28 -0.29 -0.66 -0.21 -0.57 0.10 -0.07 -0.57 -
0.53 -0.54 0.30 -0.31 -0.47 -0.06
2101 -0.29 -0.16 -0.46 0.07 -0.02 -0.30 -0.05 -0.03 0.30 -0.14 -0.27 0.47 -
0.18 0.13 -0.24 0.29 0.14 0.00
2142 -0.30 -0.02 0.01 -0.33 -0.06 0.01 -0.39 0.09 -0.35 0.03 0.17 -0.40 0.06 -
0.56 0.48 -0.25 -0.29 0.23
2158 0.47 0.19 0.31 0.02 0.13 0.16 0.05 0.44 0.16 0.54 0.44 0.21 0.15 0.07
0.13 -0.18 -0.18 0.22
2174 -0.47 -0.81 -0.79 -0.66 -0.67 -0.65 -0.67 -0.54 -0.59 -0.08 -0.59 -0.28 -
0.16 -0.44 0.24 -0.51 -0.54 -0.59
2229 -0.14 -0.24 0.04 -0.32 0.00 -0.04 -0.09 0.08 -0.36 0.34 0.01 -0.10 -0.16 -
0.08 0.41 -0.59 -0.41 -0.11
2304 0.72 0.54 0.51 0.60 0.16 0.36 0.53 0.19 0.31 0.08 0.23 0.29 0.30 0.32 -
0.32 0.20 0.25 -0.08
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
198
Table 30 (cont.). Correlation matrix for neutral glycans derived from
embryonic stem cells.
1393 1403 1419 1444 1460 1485 1501 1540 1555 1565 1581 1590 1606 1622 1647
1663 1688 1702 1704 1717
730 -0.10 0.20 -0.50 -035 -0.29 005 0.41 009 0.16 066 -0.26 0.43 -0.16 -0.27 -
0.13 -0.54 0.46 0.22 -0.28 0.29
771 -0.36 0.58 -0.31 -007 -0.22 -018 0.63 040 0.06 053 -0.40 0.07 -0.24 -0.18 -
0.37 -0.76 0.18 0.54 -0.14 0.16
892 -0.43 0.29 -0.52 -024 -0.02 008 0.45 057 0.04 041 -0.61 0.19 -0.09 -0.05 -
0.34 -0.62 0.24 0.69 -0.06 0.18
917 -0.22 0.82 -0.01 014 -0.38 -016 0.75 006 0.08 065 -0.19 -0.02 -0.12 -0.31 -
0.21 -0.72 0.36 0.24 -0.05 0.02
933 -0.26 0.65 -0.02 009 -0.12 -029 0.53 045 0.13 020 -0.44 -0.02 -0.05 -0.07 -
0.28 -0.63 -0.12 0.57 0.12 0.12
1054 -0.57 0.32 -0.42 -005 -0.05 -005 0.44 053 -0.22 032 -0.60 0.21 -0.11 -
0.08 -0.30 -0.46 0.12 0.65 -0.06 -0.16
1079 -0.24 0.84 -0.05 017 -0.19 004 0.69 006 0.09 059 -0.34 -0.08 0.15 -0.14 -
0.04 -0.62 0.39 0.26 0.21 -0.01
1095 -0.10 0.52 0.08 004 -0.10 -021 0.20 054 0.08 018 -0.36 0.18 0.10 -0.07 -
0.14 -0.50 -0.02 0.65 0.04 0.37
1120 -0.25 0.82 -0.22 050 -0.53 -042 0.83 019 -0.24 028 -0.19 0.07 -0.31 -0.44
-0.43 -0.45 0.14 0.35 -0.22 -0.04
1136 -0.25 -0.29 -0.67 -006 0.16 018 -0.21 029 -0.22 -0.17 -0.41 0.33 0.12
0.11 0.13 0.36 0.05 0.38 0.01 0.26
1216 -0.16 0.15 -0.21 -016 -0.09 -018 0.13 0.84 0.12 024 -0.51 0.45 -0.20 -
0.08 -0.39 -0.55 -0.07 0.89 -0.22 0.26
1241 -0.01 0.84 0.25 058 -0.54 -028 0.47 -002 0.00 035 0.06 0.14 0.07 -0.49
0.00 -0.44 0.22 0.20 -0.04 0.04
1257 0.24 0.32 0.22 -003 0.37 037 -0.07 -005 -0.04 043 -0.17 -0.06 0.48 0.42
0.44 -0.04 0.40 -0.05 0.34 -0.10
1282 -0.29 0.76 -0.28 051 -0.39 -003 0.72 -009 -0.28 030 -0.18 -0.03 0.07 -
0.34 -0.10 -0.21 0.36 0.12 0.08 -0.08
1298 0.08 -0.57 -0.12 -032 0.75 039 -0.75 017 -0.02 -029 -0.28 -0.01 0.41 0.66
0.48 0.42 -0.15 0.11 0.41 0.01
1323 -0.48 0.73 -0.08 054 -0.47 -041 0.79 005 -0.39 014 -0.06 -0.21 -0.39 -
0.38 -0.51 -0.28 -0.04 0.12 -0.19 -0.22
1339 -0.31 0.73 -0.14 031 -0.40 -036 0.95 -002 -0.23 017 -0.11 -0.22 -0.34 -
0.33 -0.51 -0.42 0.10 0.05 -0.15 -0.24
1378 -0.20 0.32 -0.15 016 -0.08 -042 0.26 0.86 -0.06 -001 -0.51 0.20 -0.32 -
0.02 -0.50 -0.45 -0.33 0.91 -0.13 0.06
1393 1.00 -0.18 0.30 -0 02 -0.04 0 01 -0.39 -0 23 0.42 0 12 0.31 0.33 0.12
0.04 0.39 0.05 0.18 -0.25 -0.07 0.17
1403 -0.18 1.00 0.12 044 -0.39 -020 0.73 -005 -0.13 044 -0.08 -0.17 0.05 -0.28
-0.13 -0.46 0.27 0.12 0.09 0.05
1419 0.30 0.12 1.00 010 -0.40 -028 -0.22 -033 0.28 000 0.78 -0.09 -0.03 -0.41
0.02 -0.16 -0.10 -0.40 -0.23 0.15
1444 -0.02 0.44 0.10 1.00 -0.22 -025 0.29 -004 -0.24 -0 16 0.09 -0.20 -0.07 -
0.18 -0.06 0.08 -0.17 0.03 0.06 -0.53
1460 -0.04 -0.39 -0.40 -0 22 1.00 0.62 -0.42 0 16 -0.22 -024 -0.58 -0.35 0.52
0.97 0.42 0.49 -0.04 0.08 0.70 -0.26
1485 0.01 -0.20 -0.28 -025 0.62 1.00 -0.33 -027 0.07 032 -0.29 -0.19 0.80 0.55
0.69 0.27 0.60 -0.20 0.65 0.02
1501 -0.39 0.73 -0.22 029 -0.42 -033 1.00 003 -0.07 024 -0.20 -0.19 -0.35 -
0.34 -0.53 -0.52 0.04 0.11 -0.12 -0.20
1540 -0.23 -0.05 -0.33 -004 0.16 -027 0.03 1.00 -0.10 -008 -0.55 0.25 -0.36
0.18 -0.53 -0.28 -0.36 0.94 -0.23 0.02
1555 0.42 -0.13 0.28 -0 24 -0.22 0 07 -0.07 -0 10 1.00 0 25 0.23 0.27 0.03 -
0.24 0.07 -0.47 0.00 -0.11 -0.05 0.33
1565 0.12 0.44 0.00 -016 -0.24 032 0.24 -008 0.25 1.00 -0.01 0.21 0.14 -0.18
0.17 -0.58 0.76 0.07 -0.11 0.26
1581 0.31 -0.08 0.78 009 -0.58 -029 -0.20 -055 0.23 -001 1.00 -0.02 -0.22 -
0.61 -0.04 -0.04 0.00 -0.63 -0.48 0.15
1590 0.33 -0.17 -0.09 -020 -0.35 -019 -0.19 025 0.27 021 -0.02 1.00 -0.14 -
0.36 0.03 -0.11 0.11 0.31 -0.44 0.31
1606 0.12 0.05 -0.03 -0 07 0.52 0.80 -0.35 -0 36 0.03 0 14 -0.22 -0.14 1.00 0
47 0.87 0.36 0.45 -0.24 0.81 0.13
1622 0.04 -0.28 -0.41 -018 0.97 0.55 -0.34 018 -0.24 -0 18 -0.61 -0.36 0.47
1.00 0.41 0.47 -0.05 0.11 0.70 -0.24
1647 0.39 -0.13 0.02 -006 0.42 069 -0.53 -053 0.07 017 -0.04 0.03 0.87 041
1.00 0.49 0.48 -0.40 0.64 0.09
1663 0.05 -0.46 -0.16 008 0.49 027 -0.52 -028 -0.47 -058 -0.04 -0.11 0.36 0.47
0.49 1.00 -0.12 -0.33 0.38 -0.21
1688 0.18 0.27 -0.10 -017 -0.04 060 0.04 -036 0.00 0.76 0.00 0.11 0.45 -0.05
0.48 -0.12 1.00 -0.17 0.10 0.25
1702 -0.25 0.12 -0.40 003 0.08 -020 0.11 0.94 -0.11 007 -0.63 0.31 -0.24 0.11 -
0.40 -0.33 -0.17 1.00 -0.14 0.10
1704 -0.07 0.09 -0.23 006 0.70 0.65 -0.12 -023 -0.05 -011 -0.48 -0.44 0.81 070
0.64 0.38 0.10 -0.14 1.00 -0.16
1717 0.17 0.05 0.15 -053 -0.26 002 -0.20 002 0.33 026 0.15 0.31 0.13 -0.24
0.09 -0.21 0.25 0.10 -0.16 1.00
1743 0.16 -0.59 0.23 -016 -0.16 -008 -0.35 -046 0.03 -029 0.69 -0.08 -0.29 -
0.21 -0.07 0.35 -0.15 -0.64 -0.38 -0.03
1768 -0.24 0.17 -0.31 -003 0.17 034 0.21 -017 -0.01 022 -0.22 -0.07 0.34 0.23
0.23 0.14 0.12 -0.18 0.28 0.05
1793 -0.20 0.57 -0.15 038 -0.52 -069 0.76 023 -0.09 -006 -0.16 0.08 -0.58 -
0.39 -0.59 -0.31 -0.30 0.30 -0.28 -0.05
1809 0.54 -0.42 0.23 030 0.21 008 -0.48 -050 -0.03 -043 0.34 -0.12 0.16 0.21
0.47 0.66 -0.12 -0.55 0.22 -0.40
1825 -0.27 0.33 -0.35 -016 -0.08 027 0.66 -018 0.00 050 -0.14 -0.27 -0.08 -
0.04 -0.24 -0.36 0.44 -0.17 0.00 -0.07
1850 0.07 0.28 -0.45 -008 0.50 0.71 0.07 -017 -0.10 043 -0.55 -0.11 0.72 0.58
0.62 0.19 0.57 -0.03 0.70 0.07
1866 -0.15 0.03 -0.37 -013 0.77 065 0.02 -008 -0.08 -006 -0.60 -0.51 0.62 0.79
0.39 0.28 0.07 -0.08 0.89 -0.19
1905 -0.16 -0.68 -0.19 -021 0.09 -015 -0.23 000 -0.06 -069 0.13 -0.14 -0.32
0.04 -0.29 0.43 -0.55 -0.18 -0.15 -0.02
1955 0.55 -0.51 0.19 005 -0.21 -017 -0.40 -047 0.13 -020 0.57 0.26 -0.25 -0.20
0.19 0.42 -0.08 -0.55 -0.34 -0.10
1996 -0.10 0.79 -0.10 044 -0.48 -028 0.84 000 -0.16 041 -0.08 -0.12 -0.29 -
0.34 -0.37 -0.43 0.27 0.11 -0.19 -0.10
2012 0.14 -0.05 -0.27 -009 0.57 055 -0.36 000 0.08 -007 -0.53 0.13 0.83 0.55
0.73 0.35 0.12 0.13 0.78 0.14
2028 0.46 -0.56 0.41 005 0.00 006 -0.65 -053 0.16 -043 0.54 0.07 0.24 -0.07
0.47 0.52 -0.16 -0.61 0.02 -0.04
2041 0.29 -0.47 0.06 -015 0.08 028 -0.59 -026 0.37 -032 0.15 0.33 0.50 -0.03
0.54 0.37 -0.04 -0.22 0.24 0.33
2067 -0.26 -0.61 0.20 -0 28 0.13 -0 12 -0.43 0 10 0.08 -0 67 0.23 -0.20 -0.13
0.02 -0.24 0.26 -0.64 -0.09 -0.05 0.13
2101 0.32 0.22 0.40 063 -0.53 -060 0.06 -024 -0.04 -024 0.47 0.13 -0.34 -0.48 -
0.05 0.09 -0.19 -0.18 -0.31 -0.17
2142 -0.19 -0.37 0.07 -017 0.35 -015 -0.37 055 -0.27 -027 0.01 -0.18 -0.30
0.31 -0.34 0.05 -0.39 0.34 -0.25 -0.07
2158 0.36 0.02 -0.28 -018 -0.10 002 -0.16 026 0.14 026 -0.26 0.77 0.16 -0.06
0.23 -0.05 0.27 0.36 -0.17 0.51
2174 0.53 -0.57 0.42 0 04 -0.03 0 01 -0.63 -0 55 0.11 -0 41 0.58 0.08 0.15 -
0.09 0.44 0.54 -0.11 -0.62 -0.04 -0.10
2229 0.06 -0.31 -0.15 -022 0.32 046 -0.38 -015 0.27 -028 -0.23 0.07 0.65 0.21
0.54 0.31 0.05 -0.03 0.57 0.21
2304 -0.06 0.37 -0.09 -023 -0.33 028 0.31 -008 0.21 0.87 -0.01 0.27 0.06 -0.32
0.05 -0.52 0.77 0.06 -0.25 0.36
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
199
Table 30 (cont.). Correlation matrix for neutral glycans derived from
embryonic stem cells.
1743 1768 1793 1809 1825 1850 1866 1905 1955 1996 2012 2028 2041 2067 2101
2142 2158 2174 2229 2304
730 -0.19 0.25 0.21 -0.60 0.54 0.24 -0.16 -0.24 -0.14 0.32 -0.09 -0.46 -0.15 -
0.46 -0.29 -0.30 0.47 -0.47 -0.14 0.72
771 -0.62 0.07 0.44 -0.79 0.37 0.11 -0.08 -0.51 -0.63 0.46 -0.10 -0.79 -0.47 -
0.46 -0.16 -0.02 0.19 -0.81 -0.24 0.54
892 -0.62 0.02 0.21 -0.84 0.38 0.16 0.06 -0.31 -0.73 0.26 0.07 -0.74 -0.24 -
0.26 -0.46 0.01 0.31 -0.79 0.04 0.51
917 -0.62 0.05 0.51 -0.56 0.44 0.20 -0.05 -0.73 -0.51 0.69 -0.15 -0.69 -0.57 -
0.68 0.07 -0.33 0.02 -0.66 -0.32 0.60
933 -0.85 -0.05 0.48 -0.62 0.01 0.06 0.08 -0.55 -0.76 0.36 0.19 -0.61 -0.29 -
0.28 -0.02 -0.06 0.13 -0.67 0.00 0.16
1054 -0.65 0.01 0.25 -0.68 0.21 0.08 0.00 -0.38 -0.66 0.25 0.08 -0.63 -0.33 -
0.29 -0.30 0.01 0.16 -0.65 -0.04 0.36
1079 -0.78 0.12 0.39 -0.54 0.40 0.37 0.17 -0.78 -0.67 0.61 0.12 -0.66 -0.42 -
0.66 -0.05 -0.39 0.05 -0.67 -0.09 0.53
1095 -0.82 -0.06 0.25 -0.62 -0.24 0.05 -0.07 -0.56 -0.73 0.18 0.31 -0.43 -0.05
-0.21 -0.03 0.09 0.44 -0.54 0.08 0.19
1120 -0.48 0.04 0.79 -0.45 0.36 0.06 -0.22 -0.39 -0.37 0.86 -0.24 -0.61 -0.47 -
0.57 0.30 -0.35 0.16 -0.59 -0.36 0.31
1136 -0.05 0.25 -0.03 -0.17 -0.17 0.16 -0.07 0.29 -0.01 -0.20 0.33 0.00 0.41
0.10 -0.14 0.03 0.54 -0.08 0.34 0.08
1216 -0.65 -0.19 0.24 -0.67 -0.09 -0.06 -0.13 -0.26 -0.58 0.12 0.12 -0.54 -
0.13 -0.07 -0.27 0.17 0.44 -0.59 0.01 0.23
1241 -0.59 -0.07 0.44 -0.26 -0.07 0.04 -0.28 -0.73 -0.35 0.57 0.06 -0.25 -0.13
-0.57 0.47 -0.40 0.21 -0.28 -0.10 0.29
1257 -0.45 0.24 -0.30 -0.02 0.04 0.51 0.31 -0.74 -0.30 0.07 0.33 -0.14 -0.25 -
0.53 -0.18 0.06 0.15 -0.16 -0.16 0.30
1282 -0.39 0.20 0.53 -0.30 0.46 0.33 0.03 -0.35 -0.34 0.77 -0.02 -0.45 -0.24 -
0.54 0.13 -0.56 0.07 -0.44 -0.08 0.32
1298 -0.05 -0.08 -0.64 0.23 -0.57 0.10 0.29 0.07 -0.02 -0.83 0.56 0.30 0.40
0.30 -0.24 0.48 0.13 0.24 0.41 -0.32
1323 -0.22 0.15 0.77 -0.34 0.41 -0.02 -0.11 -0.16 -0.25 0.82 -0.44 -0.50 -0.64
-0.31 0.29 -0.25 -0.18 -0.51 -0.59 0.20
1339 -0.30 0.08 0.69 -0.38 0.61 0.03 -0.03 -0.27 -0.36 0.83 -0.40 -0.59 -0.62 -
0.47 0.14 -0.29 -0.18 -0.54 -0.41 0.25
1378 -0.69 -0.30 0.49 -0.51 -0.21 -0.15 -0.08 -0.23 -0.57 0.25 0.05 -0.57 -
0.30 -0.06 0.00 0.23 0.22 -0.59 -0.11 -0.08
1393 0.16 -0.24 -0.20 0.54 -0.27 0.07 -0.15 -0.16 0.55 -0.10 0.14 0.46 0.29 -
0.26 0.32 -0.19 0.36 0.53 0.06 -0.06
1403 -0.59 0.17 0.57 -0.42 0.33 0.28 0.03 -0.68 -0.51 0.79 -0.05 -0.56 -0.47 -
0.61 0.22 -0.37 0.02 -0.57 -0.31 0.37
1419 0.23 -0.31 -0.15 0.23 -0.35 -0.45 -0.37 -0.19 0.19 -0.10 -0.27 0.41 0.06
0.20 0.40 0.07 -0.28 0.42 -0.15 -0.09
1444 -0.16 -0.03 0.38 0.30 -0.16 -0.08 -0.13 -0.21 0.05 0.44 -0.09 0.05 -0.15 -
0.28 0.63 -0.17 -0.18 0.04 -0.22 -0.23
1460 -0.16 0.17 -0.52 0.21 -0.08 0.50 0.77 0.09 -0.21 -0.48 0.57 0.00 0.08
0.13 -0.53 0.35 -0.10 -0.03 0.32 -0.33
1485 -0.08 0.34 -0.69 0.08 0.27 0.71 0.65 -0.15 -0.17 -0.28 0.55 0.06 0.28 -
0.12 -0.60 -0.15 0.02 0.01 0.46 0.28
1501 -0.35 0.21 0.76 -0.48 0.66 0.07 0.02 -0.23 -0.40 0.84 -0.36 -0.65 -0.59 -
0.43 0.06 -0.37 -0.16 -0.63 -0.38 0.31
1540 -0.46 -0.17 0.23 -0.50 -0.18 -0.17 -0.08 0.00 -0.47 0.00 0.00 -0.53 -0.26
0.10 -0.24 0.55 0.26 -0.55 -0.15 -0.08
1555 0.03 -0.01 -0.09 -0.03 0.00 -0.10 -0.08 -0.06 0.13 -0.16 0.08 0.16 0.37
0.08 -0.04 -0.27 0.14 0.11 0.27 0.21
1565 -0.29 0.22 -0.06 -0.43 0.50 0.43 -0.06 -0.69 -0.20 0.41 -0.07 -0.43 -0.32
-0.67 -0.24 -0.27 0.26 -0.41 -0.28 0.87
1581 0.69 -0.22 -0.16 0.34 -0.14 -0.55 -0.60 0.13 0.57 -0.08 -0.53 0.54 0.15
0.23 0.47 0.01 -0.26 0.58 -0.23 -0.01
1590 -0.08 -0.07 0.08 -0.12 -0.27 -0.11 -0.51 -0.14 0.26 -0.12 0.13 0.07 0.33 -
0.20 0.13 -0.18 0.77 0.08 0.07 0.27
1606 -0.29 0.34 -0.58 0.16 -0.08 0.72 0.62 -0.32 -0.25 -0.29 0.83 0.24 0.50 -
0.13 -0.34 -0.30 0.16 0.15 0.65 0.06
1622 -0.21 0.23 -0.39 0.21 -0.04 0.58 0.79 0.04 -0.20 -0.34 0.55 -0.07 -0.03
0.02 -0.48 0.31 -0.06 -0.09 0.21 -0.32
1647 -0.07 0.23 -0.59 0.47 -0.24 0.62 0.39 -0.29 0.19 -0.37 0.73 0.47 0.54 -
0.24 -0.05 -0.34 0.23 0.44 0.54 0.05
1663 0.35 0.14 -0.31 0.66 -0.36 0.19 0.28 0.43 0.42 -0.43 0.35 0.52 0.37 0.26
0.09 0.05 -0.05 0.54 0.31 -0.52
1688 -0.15 0.12 -0.30 -0.12 0.44 0.57 0.07 -0.55 -0.08 0.27 0.12 -0.16 -0.04 -
0.64 -0.19 -0.39 0.27 -0.11 0.05 0.77
1702 -0.64 -0.18 0.30 -0.55 -0.17 -0.03 -0.08 -0.18 -0.55 0.11 0.13 -0.61 -
0.22 -0.09 -0.18 0.34 0.36 -0.62 -0.03 0.06
1704 -0.38 0.28 -0.28 0.22 0.00 0.70 0.89 -0.15 -0.34 -0.19 0.78 0.02 0.24 -
0.05 -0.31 -0.25 -0.17 -0.04 0.57 -0.25
1717 -0.03 0.05 -0.05 -0.40 -0.07 0.07 -0.19 -0.02 -0.10 -0.10 0.14 -0.04 0.33
0.13 -0.17 -0.07 0.51 -0.10 0.21 0.36
1743 1.00 -0.06 -0.29 0.47 0.02 -0.44 -0.31 0.68 0.76 -0.33 -0.51 0.58 0.19
0.49 0.13 0.17 -0.32 0.63 -0.17 -0.21
1768 -0.06 1.00 -0.02 -0.25 0.41 0.56 0.34 -0.03 -0.14 0.16 0.26 -0.12 0.03 -
0.15 -0.41 -0.19 0.20 -0.26 -0.04 0.29
1793 -0.29 -0.02 1.00 -0.26 0.18 -0.16 -0.21 -0.03 -0.10 0.72 -0.34 -0.48 -
0.47 -0.27 0.44 -0.31 0.03 -0.44 -0.39 0.01
1809 0.47 -0.25 -0.26 1.00 -0.41 -0.06 0.06 0.25 0.74 -0.31 0.10 0.73 0.31
0.08 0.49 -0.13 -0.25 0.83 0.18 -0.55
1825 0.02 0.41 0.18 -0.41 1.00 0.38 0.30 -0.06 -0.24 0.58 -0.33 -0.53 -0.50 -
0.33 -0.46 -0.28 -0.20 -0.52 -0.31 0.54
1850 -0.44 0.56 -0.16 -0.06 0.38 1.00 0.73 -0.39 -0.29 0.20 0.63 -0.27 -0.01 -
0.53 -0.45 -0.39 0.26 -0.30 0.23 0.32
1866 -0.31 0.34 -0.21 0.06 0.30 0.73 1.00 0.01 -0.39 -0.08 0.58 -0.18 -0.01
0.00 -0.57 -0.15 -0.25 -0.23 0.37 -0.19
1905 0.68 -0.03 -0.03 0.25 -0.06 -0.39 0.01 1.00 0.41 -0.36 -0.23 0.38 0.27
0.80 -0.10 0.20 -0.27 0.36 0.14 -0.53
1955 0.76 -0.14 -0.10 0.74 -0.24 -0.29 -0.39 0.41 1.00 -0.22 -0.28 0.68 0.26
0.10 0.50 -0.14 0.00 0.78 -0.12 -0.21
1996 -0.33 0.16 0.72 -0.31 0.58 0.20 -0.08 -0.36 -0.22 1.00 -0.38 -0.54 -0.63 -
0.62 0.23 -0.43 0.00 -0.50 -0.52 0.42
2012 -0.51 0.26 -0.34 0.10 -0.33 0.63 0.58 -0.23 -0.28 -0.38 1.00 0.15 0.59 -
0.08 -0.28 -0.25 0.40 0.05 0.76 -0.16
2028 0.58 -0.12 -0.48 0.73 -0.53 -0.27 -0.18 0.38 0.68 -0.54 0.15 1.00 0.69
0.41 0.33 -0.11 -0.01 0.96 0.36 -0.41
2041 0.19 0.03 -0.47 0.31 -0.50 -0.01 -0.01 0.27 0.26 -0.63 0.59 0.69 1.00
0.39 0.00 -0.23 0.36 0.59 0.81 -0.24
2067 0.49 -0.15 -0.27 0.08 -0.33 -0.53 0.00 0.80 0.10 -0.62 -0.08 0.41 0.39
1.00 -0.20 0.41 -0.34 0.32 0.28 -0.57
2101 0.13 -0.41 0.44 0.49 -0.46 -0.45 -0.57 -0.10 0.50 0.23 -0.28 0.33 0.00 -
0.20 1.00 -0.22 -0.01 0.42 -0.22 -0.23
2142 0.17 -0.19 -0.31 -0.13 -0.28 -0.39 -0.15 0.20 -0.14 -0.43 -0.25 -0.11 -
0.23 0.41 -0.22 1.00 -0.18 -0.11 -0.31 -0.29
2158 -0.32 0.20 0.03 -0.25 -0.20 0.26 -0.25 -0.27 0.00 0.00 0.40 -0.01 0.36 -
0.34 -0.01 -0.18 1.00 -0.09 0.15 0.37
2174 0.63 -0.26 -0.44 0.83 -0.52 -0.30 -0.23 0.36 0.78 -0.50 0.05 0.96 0.59
0.32 0.42 -0.11 -0.09 1.00 0.31 -0.42
2229 -0.17 -0.04 -0.39 0.18 -0.31 0.23 0.37 0.14 -0.12 -0.52 0.76 0.36 0.81
0.28 -0.22 -0.31 0.15 0.31 1.00 -0.24
2304 -0.21 0.29 0.01 -0.55 0.54 0.32 -0.19 -0.53 -0.21 0.42 -0.16 -0.41 -0.24 -
0.57 -0.23 -0.29 0.37 -0.42 -0.24 1.00
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
200
Table 31. Correlation matrix for acidic glycans derived from embryonic stem
cells.
1354 1362 1403 1475 1500 1516 1541 1549 1557 1565 1637 1678 1703 1711 1719
1727 1744 1768 1791 1799
1354 1.00 0.00 -0.15 0.11 063 0.75 0.91 -0.18 0.37 0.25 0.13 0.27 0.46 -0.54
061 0.05 0.46 0.33 0.91 0.12
1362 0.00 1.00 0.47 -016 -021 0.13 0.09 0.03 0.06 0.22 -0.20 -0.11 0.04 0.12
0.11 0.02 0.07 0.37 -0.11 0.00
1403 -0.15 0.47 1.00 -014 -014 0.09 0.05 0.19 0.30 0.11 -0.16 -0.20 0.21 -0.16
017 005 0.06 0.28 -0.02 -0.06
1475 0.11 -0 .16 -0.14 1.00 056 0.11 0.12 -0.23 -0.17 -0.18 0.95 0.70 0.34
0.32 -001 0.29 0.41 -0.21 0.21 0.78
1500 0.63 -0.21 -0.14 056 1.00 0.52 0.65 -0.15 -0.04 0.16 0.65 0.58 0.69 -0.14
057 0.09 0.47 -0.04 0.74 0.35
1516 0.75 0.13 0.09 0.11 0.52 1.00 0.63 -0.13 -0.04 0.33 0.11 0.28 0.36 -0.30
048 0.07 0.71 0.56 0.77 -0.08
1541 0.91 0.09 0.05 012 0.65 0.63 1.00 -0.13 0.39 0.17 0.14 0.14 0.52 -0.56
056 -0.05 0.45 0.25 0.92 0.21
1549 -0.18 0.03 0.19 -023 -0.15 -0.13 -0.13 1.00 -0.03 0.26 -0.26 -0.20 -0.20 -
0.03 -020 0.17 -0.15 -0.09 -0.12 -0.16
1557 0.37 0.08 0.30 -017 -0.04 -0.04 0.39 -0.03 1.00 0.19 -0.20 0.05 0.31 -
0.46 048 0.22 -0.20 0.22 0.21 0.09
1565 0.25 0.22 0.11 -018 0.16 0.33 0.17 0.26 0.19 1.00 -0.14 0.33 0.27 -0.17
025 0.56 0.00 0.32 0.17 -0.03
1637 0.13 -0.20 -0.16 0.95 065 0.11 0.14 -0.26 -0.20 -0.14 1.00 0.73 0.32 0.34
005 0.28 0.46 -0.25 0.24 0.74
1678 0.27 -0 .11 -0.20 070 058 0.28 0.14 -0.20 0.05 0.33 0.73 1.00 0.47 0.11
029 0.69 0.41 0.25 0.18 0.58
1703 0.46 0.04 0.21 034 069 0.36 0.52 -0.20 0.31 0.27 0.32 0.47 1.00 -0.14
0.80 0.06 0.15 0.26 0.47 0.27
1711 -0.54 0.12 -0.16 032 -014 -0.30 -0.56 -0.03 -0.46 -0.17 0.34 0.11 -0.14
1.00 -028 -0.08 -0.07 -0.27 -0.49 0.16
1719 0.61 0.11 0.17 -001 057 0.48 0.56 -0.20 0.48 0.25 0.05 0.29 0.60 -0.28
1.00 -0.10 0.17 0.35 0.53 -0.13
1727 005 0.02 0 05 029 009 007 -0.05 0.17 022 056 028 0.69 0.06 -0 08 -0.10
1.00 003 0.24 -0.06 0.36
1744 0.46 0.07 0.06 041 047 0.71 0.45 -0.15 -0.20 0.00 0.46 0.41 0.15 -0.07
0.17 0.03 1.00 0.48 0.51 0.36
1768 0.33 0.37 0.28 -021 -0.04 0.56 0.25 -0.09 0.22 0.32 -0.25 0.25 0.26 -0.27
0.35 024 0.48 1.00 0.13 -0.02
1791 0.91 -0 .11 -0.02 021 0.74 0.77 0.92 -0.12 0.21 0.17 0.24 0.18 0.47 -0.49
0.53 -0.06 0.51 0.13 1.00 0.10
1799 0.12 0.00 -0.06 0.78 035 -0.08 0.21 -0.16 0.09 -0.03 0.74 0.58 0.27 0.16 -
0.13 0.36 0.36 -0.02 0.10 1.00
1840 -0.13 -0 .06 -0.12 046 0.17 -0.04 -0.20 -0.25 -0.13 0.29 0.52 0.67 0.09
0.25 -0.14 048 0.33 0.20 -0.21 0.63
1865 0.65 0.23 0.51 016 055 0.46 0.75 -0.08 0.68 0.33 0.18 0.29 0.71 -0.47
0.74 0.17 0.28 0.30 0.66 0.24
1873 0.17 0.01 0.02 047 035 0.24 0.11 -0.14 0.22 0.21 0.46 0.81 0.35 -0.03
0.29 0.58 0.44 0.48 0.05 0.42
1889 0.23 0.04 0.13 022 025 0.25 0.11 0.03 0.43 0.52 0.18 0.72 0.45 -0.27 0.44
0.69 0.17 0.50 0.07 0.20
1906 0.42 0.25 0.07 058 0.73 0.40 0.44 -0.20 0.07 0.26 0.68 0.77 0.59 0.08
0.48 0.45 0.50 0.25 0.40 0.51
1914 0.09 0.27 0.23 040 048 0.30 0.13 0.07 0.02 0.36 0.46 0.64 0.39 0.21 037
047 0.39 0.25 0.13 0.19
1930 -0.25 0.14 0.26 -0.74 -0.74 -0.17 -0.24 0.36 0.24 0.30 -0.81 -0.56 -0.51 -
0.36 -031 0.01 -0.32 0.18 -0.31 -0.47
1946 -0.38 0.07 0.41 -042 -045 -0.09 -0.26 0.44 -0.06 0.32 -0.48 -0.35 -0.33 -
0.32 -0.31 009 -0.09 0.08 -0.25 -0.33
1947 0.47 -0 .01 -0.29 058 0.73 0.27 0.35 -0.36 0.04 0.02 0.71 0.74 0.46 0.10
047 0.29 0.34 0.02 0.37 0.42
2002 0.07 -0 .05 -0.05 057 052 0.28 -0.04 -0.37 -0.22 0.17 0.62 0.82 0.40 0.16
022 0.47 0.41 0.28 0.04 0.37
2010 0.81 0.1 fi 0.09 024 045 0.60 0.78 -0.19 0.58 0.20 0.26 0.35 0.35 -0.37
056 0.15 0.56 0.35 0.72 0.30
2011 -0.15 -0.24 -0.17 008 033 -0.16 -0.12 -0.21 -0.30 -0.17 0.20 0.14 0.30 -
0.03 029 -0.34 0.06 -0.15 -0.10 -0.02
2018 0.18 -0 .09 -0 06 -020 -024 008 -0.14 -0.08 052 022 -022 0.26 0.03 -0 20
028 037 -016 0 40 -0.12 -0.15
2035 0.19 0.00 -0.04 058 049 0.45 0.10 -0.24 -0.18 0.46 0.65 0.74 0.35 0.37
016 0.47 0.54 0.23 0.23 0.48
2052 0.10 -0 .14 -0.24 050 066 0.05 0.05 0.00 -0.11 0.39 0.56 0.76 0.56 0.22
031 0.50 0.02 -0.06 0.09 0.33
2068 0.62 0.01 -0.06 061 0.76 0.56 0.60 -0.25 0.05 0.29 0.70 0.69 0.51 -0.03
035 0.30 0.70 0.24 0.62 0.67
2076 -0.46 0.23 0.10 -0.77 -0.84 -0.37 -0.43 0.38 0.04 0.10 -0.80 -0.71 -0.66
0.03 -044 -0.19 -0.44 -0.02 -0.52 -0.54
2092 -0.53 0.02 -0.06 -058 -051 -0.36 -0.52 0.27 -0.30 0.15 -0.57 -0.43 -0.49 -
0.12 -035 -010 -0.42 -0.16 -0.50 -0.58
2117 -0.51 -0 .13 -0.32 031 -007 -0.27 -0.57 0.02 -0.51 -0.38 0.36 0.25 -0.31
0.71 -031 0.06 0.08 -0.17 -0.48 0.01
2133 0.32 0.19 0.15 064 030 0.27 0.34 -0.26 0.26 0.20 0.56 0.66 0.32 -0.09 004
068 0.33 0.25 0.27 0.69
2156 0.81 0.10 0.16 024 066 0.53 0.79 -0.12 0.64 0.41 0.29 0.48 0.63 -0.46
0.75 0.31 0.32 0.25 0.75 0.26
2157 0.01 -0 .28 -0.20 000 -007 -0.07 -0.17 -0.24 0.05 -0.16 0.02 0.12 0.11
0.00 009 -006 -0.06 0.16 -0.14 0.10
2164 0.07 0.18 0.04 -030 010 0.24 0.13 -0.16 -0.22 0.34 -0.17 0.06 0.15 -0.19
0.16 0.00 0.28 0.43 0.05 -0.11
2221 -0.23 0.07 0.22 -029 -048 -0.15 -0.23 -0.16 0.06 -0.18 -0.31 -0.44 -0.33
0.13 -0.26 -0.21 -0.20 -0.03 -0.22 -0.20
2222 -0.37 -0 .23 -0.22 -039 -037 -0.37 -0.37 0.40 -0.33 -0.13 -0.41 -0.52 -
0.47 -0.06 -0.42 -0.31 -0.37 -0.37 -0.31 -0.38
2230 0.60 0.10 0.17 024 068 0.41 0.66 -0.22 0.43 0.10 0.35 0.38 0.71 -0.21
0.85 -0.12 0.46 0.24 0.58 0.22
2237 -0.33 0.27 0.54 -040 -048 -0.15 -0.27 0.23 0.10 0.15 -0.45 -0.29 -0.35 -
0.44 -0.26 026 -0.26 0.10 -0.30 -0.37
2238 -0.14 -0 .21 -0.12 015 026 -0.16 -0.10 0.05 -0.17 -0.20 0.20 -0.02 0.22
0.24 0.18 -038 -0.05 -0.31 -0.04 0.00
2239 -0.10 0.32 -0.17 007 026 -0.26 -0.04 -0.21 -0.05 0.03 0.16 0.21 0.36 0.34
0.35 -003 -0.31 -0.18 -0.18 -0.02
2246 059 -0 .09 -0 06 025 054 0.70 0 46 -0.08 -0.11 0.20 028 0.13 018 -0 09
0.32 -004 035 -0.16 0.74 -0.15
2253 -0.24 0.33 0.64 -036 -0.38 -0.09 -0.13 0.03 0.14 0.20 -0.41 -0.34 -0.15 -
0.46 -0.11 002 -0.16 0.10 -0.18 -0.22
2254 -0.19 0.08 0.29 -024 -034 -0.11 -0.12 -0.01 0.00 0.04 -0.27 -0.28 -0.17 -
0.37 -0.17 -004 -0.14 -0.05 -0.12 -0.16
2263 -0.43 -0 .29 -0.32 017 003 -0.34 -0.43 0.53 -0.56 -0.11 0.17 0.02 -0.22
0.32 -0.38 004 -0.21 -0.48 -0.31 -0.06
2279 0.04 0.17 -0.04 042 009 0.02 0.05 -0.33 -0.01 -0.30 0.41 0.23 0.05 0.50 -
0.07 007 0.19 0.03 0.00 0.40
2280 0.03 0.19 0.04 046 -001 0.22 -0.05 -0.14 -0.17 0.02 0.37 0.32 0.00 0.42 -
026 035 0.25 0.17 0.00 0.43
2295 -0.09 0.23 0.19 -050 -013 -0.07 0.04 0.32 0.07 0.37 -0.42 -0.10 -0.19 -
0.42 -0.08 033 -0.12 0.23 -0.12 -0.31
2321 -0.02 -0 .19 -0.13 008 019 0.05 0.00 -0.16 -0.23 -0.33 0.01 0.06 0.36 -
0.17 0.30 -034 0.07 0.10 0.01 -0.15
2367 -0.45 -0 .17 -0.10 -059 -0.75 -0.42 -0.47 0.10 -0.16 -0.37 -0.63 -0.82 -
0.70 0.09 -0.57 -0.49 -0.46 -0.27 -0.43 -0.48
2368 -0.18 -0 .19 -0.17 -033 -047 -0.16 -0.29 0.11 -0.05 -0.20 -0.33 -0.30 -
0.60 -0.20 -0.38 002 -0.19 -0.09 -0.22 -0.36
2383 -0.27 0.08 0.03 -034 -043 -0.07 -0.23 0.45 -0.17 0.27 -0.39 -0.23 -0.37 -
0.13 -0.32 010 -0.06 0.07 -0.24 -0.27
2384 -0.20 -0 .10 -0.08 -0.18 -0.09 -0.10 -0.17 -0.17 -0.24 -0.02 -0.25 -0.16 -
0.02 -0.30 -0.03 -028 -0.09 -0.02 -0.16 -0.26
2390 -0.12 -0 .01 -0.02 -044 -032 -0.11 -0.10 0.72 -0.21 0.16 -0.48 -0.42 -
0.39 -0.36 -0.37 -0.04 -0.14 -0.02 -0.11 -0.24
2400 -0.21 -0 .13 -0.09 019 0.11 -0.22 -0.20 -0.11 -0.16 -0.52 0.16 0.09 0.04
0.09 0.04 000 -0.23 -0.22 -0.17 -0.21
2408 -0.34 -0 .08 -0.10 -028 -036 -0.25 -0.32 0.43 -0.26 -0.24 -0.36 -0.48 -
0.24 0.15 -0.21 -043 -0.26 -0.19 -0.29 -0.35
2425 -0.18 -0 .30 -0.38 058 028 -0.15 -0.24 -0.19 -0.35 -0.40 0.61 0.42 0.02
0.37 -0.06 001 0.16 -0.30 -0.13 0.25
2441 -0.53 -0 .18 -0.23 -033 -052 -0.59 -0.43 0.27 -0.34 -0.48 -0.36 -0.75 -
0.58 0.34 -0.62 -0.53 -0.51 -0.57 -0.43 -0.27
2447 0.70 0.09 0.01 -014 052 0.52 0.67 -0.21 0.49 0.53 0.01 0.31 0.53 -0.31
0.74 0.14 0.26 0.38 0.60 -0.02
2448 0.46 0.17 0.20 -021 -004 0.18 0.45 -0.11 0.90 0.29 -0.25 0.17 0.32 -0.43
0.51 0.26 0.03 0.56 0.23 0.08
2482 0.19 -0 .31 -0.22 0.91 061 0.23 0.21 -0.27 -0.16 -0.25 0.88 0.70 0.34
0.21 0.08 0.21 0.57 -0.05 0.30 0.65
2512 -0.09 0.86 0.86 -017 -021 0.13 0.08 0.13 0.21 0.20 -0.21 -0.18 0.15 -0.03
0.16 004 0.08 0.38 -0.08 -0.03
2513 0.09 0.85 -0.06 -010 -015 0.09 0.07 -0.08 -0.11 0.18 -0.13 -0.01 -0.07
0.23 0.02 000 0.05 0.25 -0.12 0.04
2521 0.34 -0 .17 -0.22 020 057 0.28 0.33 -0.36 -0.06 0.50 0.21 0.39 0.60 -0.24
0.41 010 0.05 -0.01 0.38 0.12
2522 0.34 -0 .01 0.03 -005 012 -0.16 0.35 -0.14 0.89 0.16 -0.06 0.19 0.49 -
0.28 0.60 0.13 -0.33 0.07 0.15 0.13
2528 -0.15 0.40 0.27 -027 -038 -0.07 -0.09 -0.03 -0.04 0.11 -0.30 -0.28 -0.19 -
0.27 -0.15 -003 -0.11 0.05 -0.16 -0.14
2529 -0.20 0.29 0.47 -015 -024 0.26 -0.09 0.02 0.05 0.25 -0.20 0.01 0.03 0.13
0.05 0.11 0.30 0.53 -0.13 -0.16
2544 -0.19 0.09 0.31 -024 -034 -0.11 -0.12 -0.01 0.01 0.04 -0.27 -0.29 -0.17 -
0.37 -0.17 -004 -0.14 -0.05 -0.12 -0.16
2570 0.01 -0 .05 -0.12 -006 -030 -0.05 -0.06 -0.25 0.11 -0.44 -0.06 -0.09 -
0.34 0.07 -0.20 -006 0.14 0.21 -0.12 0.05
2571 0.15 -0 .15 -0.11 015 012 0.14 0.06 -0.22 -0.01 -0.48 0.12 -0.13 0.02
0.24 0.19 -036 -0.01 -0.24 0.21 -0.23
2586 -0.21 0.15 0.44 -025 014 -0.09 -0.10 0.02 0.05 0.35 -0.03 0.00 0.25 0.14
0.29 004 -0.12 0.00 -0.12 -0.16
2587 -0.24 -0 .27 -0.24 -023 -029 -0.28 -0.25 0.29 -0.25 -0.33 -0.23 -0.43 -
0.49 -0.10 -0.37 -035 -0.16 -0.34 -0.19 -0.24
2603 -0.17 -0 .01 0.09 016 018 -0.14 -0.13 -0.05 -0.06 -0.35 0.09 0.02 0.48
0.14 0.45 -042 -0.15 -0.11 -0.12 -0.16
2644 0.11 -0 .13 -0.10 036 017 -0.05 0.13 -0.31 0.10 -0.36 0.36 0.10 0.13 0.36
0.02 -012 0.09 -0.14 0.12 0.36
2645 -0.09 -0 .08 -0.08 -002 -015 -0.15 -0.11 -0.17 -0.07 -0.27 -0.05 -0.22 -
0.05 0.35 -0.18 -019 -0.23 -0.13 -0.10 0.02
2660 -0.15 -0 .09 -0.06 -020 -012 -0.15 -0.14 0.97 -0.11 0.23 -0.22 -0.15 -
0.26 0.01 -0.24 0.16 -0.16 -0.16 -0.12 -0.15
2683 0.27 -0 .06 -0.22 042 064 0.12 0.35 -0.27 0.07 0.28 0.42 0.50 0.80 0.20
0.52 0.07 0.03 -0.03 0.29 0.36
2714 -0.15 -0 .09 -0.06 058 015 -0.15 -0.14 -0.08 -0.11 0.24 0.46 0.46 0.31
0.32 -0.24 048 -0.16 -0.16 -0.12 0.61
2732 -0.12 -0 .07 -0.02 -049 -050 -0.11 -0.09 0.14 -0.11 -0.30 -0.54 -0.79 -
0.46 0.07 -0.38 -0.57 -0.21 -0.14 -0.07 -0.34
2733 -0.14 -0 .31 -0.19 -008 -009 -0.27 -0.26 -0.05 0.16 0.21 -0.04 0.11 -0.06
0.10 -0.13 036 -0.49 -0.20 -0.20 -0.05
2807 -0.28 -0 .30 -0.16 -005 -033 -0.39 -0.25 0.14 -0.15 -0.62 -0.12 -0.60 -
0.44 0.30 -0.44 -047 -0.41 -0.64 -0.14 -0.19
2878 -0.21 0.07 0.34 -002 -016 -0.14 -0.20 -0.04 0.12 0.31 -0.11 0.03 0.02 -
0.22 -0.16 029 -0.23 0.02 -0.19 0.08
2879 -0.25 0.42 0.58 -019 -032 -0.10 -0.12 0.02 0.04 -0.08 -0.21 -0.23 -0.18 -
0.26 -0.16 016 -0.14 0.06 -0.18 -0.20
2880 -0.15 -0 .09 -0.06 -020 -030 -0.15 -0.14 -0.08 -0.11 -0.36 -0.22 -0.47 -
0.26 0.12 -0.24 -043 -0.16 -0.16 -0.12 -0.15
2886 0.57 -0 .22 -0.16 044 0.80 0.42 0.56 -0.19 0.03 0.43 0.51 0.49 0.63 -0.06
0.37 0.26 0.21 -0.09 0.66 0.40
2936 -0.10 -0 .07 0.00 -027 -037 -0.12 -0.17 -0.08 0.07 -0.28 -0.29 -0.40 -
0.23 0.22 -0.14 -031 -0.20 -0.01 -0.15 -0.19
2953 -0.27 -0 .16 -0.11 -037 -045 -0.28 -0.26 0.42 -0.20 -0.09 -0.40 -0.52 -
0.47 -0.15 -0.44 -021 -0.30 -0.29 -0.22 -0.27
3024 0.54 -0 .15 -0.11 001 017 0.55 0.22 -0.13 0.24 0.30 0.01 0.23 0.09 -0.29
0.38 021 0.12 0.12 0.45 -0.25
3025 0.01 -0 .13 -0.09 -029 -039 -0.06 -0.20 -0.11 0.27 0.15 -0.32 0.01 -0.18 -
0.39 0.01 022 -0.24 0.16 -0.17 -0.22
3098 -0.17 -0 .10 -0.05 -025 -034 -0.18 -0.17 -0.09 -0.10 -0.33 -0.28 -0.50 -
0.30 0.23 -0.27 -042 -0.20 -0.16 -0.15 -0.18
3099 -0.17 -0 .16 -0.08 -038 -044 -0.32 -0.10 0.12 0.01 -0.19 -0.42 -0.61 -
0.40 -0.27 -0.36 -035 -0.35 -0.32 -0.12 -0.21
3170 -0.15 -0 .09 -0.06 009 -0.01 -0.15 -0.14 -0.08 -0.11 -0.36 0.11 0.08 -
0.26 -0.01 -0.24 029 -0.16 -0.16 -0.12 -0.15
3171 -0.15 0.47 1.00 -014 -014 0.09 0.05 0.19 0.30 0.11 -0.16 -0.20 0.21 -0.16
0.17 005 0.06 0.28 -0.02 -0.06
3172 -0.19 -0 .12 -0.08 -026 -039 -0.20 -0.19 -0.10 -0.15 -0.35 -0.29 -0.53 -
0.34 0.00 -0.32 -043 -0.22 -0.21 -0.16 -0.20
3390 -0.04 -0 .12 -0.09 -028 -038 -0.10 -0.19 -0.11 0.15 0.10 -0.30 -0.07 -
0.22 -0.39 -0.08 013 -0.23 0.05 -0.16 -0.21
3463 -021 -0 .13 -0 09 -028 -027 -022 -020 -0.11 -0.16 0.10 -031 -0.28 -036 -0
42 -034 -008 -023 -022 -0.17 -021
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
201
Table 31 (cont.). Correlation matrix for acidic glycans derived from embryonic
stem cells.
1840 1865 1873 1889 1906 1914 1930 1946 1947 2002 2010 2011 2018 2035 2052
2068 2076 2092 2117 2133 2156
1354 -0.13 D.65 0.17 0.23 042 0.09 -0.25 -0.38 0.47 0.07 0.81 -0.15 0.18 0.19
0.10 0.62 -0.46 -053 -0.51 0.32 0.81
1362 -0.06 0.23 0.01 0.04 025 0.27 0.14 0.07 -0.01 -0.05 0.16 -0.24 -0.09 D.00
-0.14 0.01 0.23 002 -0.13 0.19 0.10
1403 -0.12 0.51 0.02 0.13 007 0.23 0.26 0.41 -0.29 -0.05 0.09 -0.17 -0.06 -
0.04 -0.24 -0.06 0.10 -006 -0.32 0.15 0.16
1475 0.46 D.16 0.47 0.22 058 0.40 -0.74 -0.42 0.58 0.57 0.24 0.08 -0.20 0.58
0.50 0.61 -0.77 -058 0.31 0.64 0.24
1500 0.17 0.55 0.35 0.25 073 0.48 -0.74 -0.45 0.73 0.52 0.45 0.33 -0.24 0.49
0.66 0.76 -0.84 -051 -0.07 0.30 0.66
1516 -0.04 D.46 0.24 0.25 040 0.30 -0.17 -0.09 0.27 0.28 0.60 -0.16 0.08 0.45
0.05 0.56 -0.37 -036 -0.27 0.27 0.53
1541 -0.20 0.75 0.11 0.11 044 0.13 -0.24 -0.26 0.35 -0.04 0.78 -0.12 -0.14
0.10 0.05 0.60 -0.43 -052 -0.57 0.34 0.79
1549 -0.25 -0.08 -0.14 0.03 -0.20 0.07 0.36 0.44 -0.36 -0.37 -0.19 -0.21 -0.08
-0.24 0.00 -0.25 0.38 027 0.02 -0.26 -0.12
1557 -0.13 D.68 0.22 0.43 007 0.02 0.24 -0.06 0.04 -0.22 0.58 -0.30 0.52 -0.18
-0.11 0.05 0.04 -030 -0.51 0.26 0.64
1565 0.29 0.33 0.21 0.52 026 0.36 0.30 0.32 0.02 0.17 0.20 -0.17 0.22 0.46
0.39 0.29 0.10 015 -0.38 0.20 0.41
1637 0.52 0.18 0.46 0.18 068 0.46 -0.81 -0.48 0.71 0.62 0.26 0.20 -0.22 0.65
0.56 0.70 -0.80 -057 0.36 0.56 0.29
1678 0.67 0.29 0.81 0.72 077 0.64 -0.56 -0.35 0.74 0.82 0.35 0.14 0.26 0.74
0.76 0.69 -0.71 -043 0.25 0.66 0.48
1703 0.09 D.71 0.35 0.45 0.59 0.39 -0.51 -0.33 0.46 0.40 0.35 0.30 0.03 0.35
0.56 0.51 -0.68 -049 -0.31 0.32 0.63
1711 0.25 -0.47 -0.03 -0.27 008 0.21 -0.36 -0.32 0.10 0.16 -0.37 -0.03 -0.20
D.37 0.22 -0.03 0.03 -012 0.71 -0.09 -0.46
1719 -0.14 0.74 0.29 0.44 048 0.37 -0.31 -0.31 0.47 0.22 0.56 0.29 0.28 0.16
0.31 0.35 -0.44 -035 -0.31 0.04 0.75
1727 0.48 0.17 0.58 0.69 045 0.47 0.01 0.09 0.29 0.47 0.15 -0.34 0.37 0.47
0.50 0.30 -0.19 -010 0.06 0.68 0.31
1744 0.33 D.28 0.44 0.17 050 0.39 -0.32 -0.09 0.34 0.41 0.56 0.06 -0.16 D.54
0.02 0.70 -0.44 -0.42 0.08 0.33 0.32
1768 0.20 0.30 0.48 0.50 025 0.25 0.18 0.08 0.02 0.28 0.35 -0.15 0.40 0.23 -
0.06 0.24 -0.02 -0.16 -0.17 0.25 0.25
1791 -0.21 0.66 0.05 0.07 040 0.13 -0.31 -0.25 0.37 0.04 0.72 -0.10 -0.12 0.23
0.D9 0.62 -0.52 -050 -0.48 0.27 0.75
1799 0.63 0.24 0.42 0.20 051 0.19 -0.47 -0.33 0.42 0.37 0.30 -0.02 -0.15 D.48
0.33 0.67 -0.54 -0.58 0.01 0.69 0.26
1840 1.00 -0.05 0.60 0.44 036 0.32 -0.21 -0.12 0.35 0.72 0.03 0.20 0.16 0.68
0.49 0.53 -0.30 -016 0.18 0.35 0.04
1865 -0.05 1.00 0.32 0.45 055 0.40 -0.17 -0.12 0.35 0.14 0.75 -0.08 0.10 0.21
0.18 0.53 -0.44 -052 -0.57 0.44 0.90
1873 0.60 0.32 1.00 0.85 057 0.74 -0.28 -0.14 0.48 0.77 0.39 0.05 0.30 0.47
0.57 0.43 -0.42 -028 0.28 0.50 0.41
1889 0.44 D.45 0.85 1.00 043 0.61 0.01 0.10 0.32 0.59 0.37 0.03 0.53 0.35 0.53
0.28 -0.29 -010 -0.06 0.44 0.53
1906 0.36 0.55 0 57 0 43 1.00 0.72 -0.71 -0 45 0.84 063 0 49 014 -007 0.65
0.65 0.79 -0.74 -055 013 064 0.63
1914 0.32 0.40 0.74 0.61 072 1.00 -0.36 -0.04 0.47 0.63 0.32 0.02 -0.10 0.55
0.64 0.40 -0.34 -017 0.38 0.37 0.41
1930 -0.21 -0.17 -0.28 0.01 -071 -0.36 1.00 0.77 -0.82 -0.55 -0.15 -0.34 0.23 -
0.45 -0.59 -0.59 0.86 062 -0.38 -0.33 -0.27
1946 -0.12 -8.12 -014 0.10 -0 45 004 0.77 1.00 -0.74 -030 -026 -004 -0.16 -024
-035 -0 44 0 57 068 -022 -019 -0.29
1947 0.35 0.35 0.48 0.32 084 0.47 -0.82 -0.74 1.00 0.64 0.42 0.28 0.14 0.49
0.66 0.69 -0.79 -055 0.22 0.42 0.58
2002 0.72 D.14 0.77 0.59 063 0.63 -0.55 -0.3D 0.64 1.00 0.07 0.32 0.15 0.66
0.73 0.53 -0.64 -025 0.32 0.40 0.23
2010 003 0.75 0 39 0 37 0 49 0 32 -015 -0.26 0 42 0.07 1.00 -027 022 030 -0.82
0 63 -0 34 -059 -036 0 47 0.86
2011 0.20 -0.08 0.05 0.03 014 0.02 -0.34 -0.04 0.28 0.32 -0.27 1.00 -0.17 -
0.01 0.31 0.07 -0.35 022 0.12 -0.37 -0.08
2018 0.16 0.10 0.30 0.53 -0.07 -0.10 0.23 -0.16 0.14 0.15 0.22 -0.17 1.00 0.03
-0.01 -0.06 0.05 -0.11 -0.13 0.08 0.26
2035 0.68 0.21 0.47 0.35 065 0.55 -0.45 -0.24 0.49 0.66 0.30 -0.01 0.03 1.00
0.54 0.77 -0.51 -043 0.17 0.51 0.32
2052 0.49 0.18 0.57 0.53 065 0.64 -0.59 -0.35 0.66 0.73 -0.02 0.31 -0.01 0.54
1.00 0.45 -0.58 -0.18 0.27 0.27 0.32
2068 0.53 0.53 0.43 0.28 079 0.40 -0.59 -0.44 0.69 0.53 0.63 0.07 -0.06 0.77
0.45 1.00 -0.73 -0.68 -0.15 0.58 0.65
2076 -0.30 -0.44 -0.42 -0.29 -074 -0.34 0.86 0.57 -0.79 -0.64 -0.34 -0.35 0.05
-0.51 -0.58 -0.73 1.00 067 -0.03 -0.54 -0.52
2092 -0.16 -0.52 -0.28 -0.10 -0.55 -0.17 0.62 0.68 -0.55 -0.25 -0.59 0.22 -
0.11 -0.43 -0.18 -0.68 0.67 1,00 0.08 -0.55 -0.54
2117 0.18 -0.57 0.28 -0.06 013 0.38 -0.38 -0.22 0.22 0.32 -0.36 0.12 -0.13
0.17 0.27 -0.15 -0.03 008 1.00 -0.10 -0.48
2133 0.35 0.44 0.50 0.44 064 0.37 -0.33 -0.19 0.42 0.40 0.47 -0.37 0.08 0.51
0.27 0.58 -0.54 -055 -0.10 1.00 0.47
2158 0.04 0.90 0.41 0.53 063 0.41 -0.27 -0.29 0.58 0.23 0.88 -0.08 0.26 0.32
0.32 0.65 -0.52 -054 -0.48 0.47 1.00
2157 0.24 -0.14 -0.04 -0.01 -008 -0.39 -0.15 -0.37 0.15 0.13 -0.12 0.20 0.48
0.09 -0.01 0.09 -0.17 -006 -0.02 -0.07 -0.08
2164 0.14 0.03 -0.07 -0.01 028 0.04 0.04 0.23 0.05 0.11 -0.06 0.42 -0.17 D.22
0.02 0.23 -0.04 023 -0.15 -0.02 0.00
2221 -0.13 -0.17 -0.35 -0.33 -0.43 -0.40 0.33 0.12 -0.40 -0.29 -0.16 -0.25
0.10 -0.20 -0.51 -0.31 0.32 -014 -0.16 -0.14 -0.28
2222 -0.26 -0.55 -0.48 -0.39 -0.63 -0.44 0.40 0.34 -0.50 -0.43 -0.54 0.10 -
0.17 -0.48 -0.25 -0.54 0.52 073 0.07 -0.61 -0.55
2230 0.05 0.78 0.38 0.34 064 0.46 -0.46 -0.36 0.59 0.26 0.72 0.35 0.02 0.27
0.29 0.60 -0.55 -051 -0.21 0.17 0.79
2237 -0.17 -0.01 -0.03 0.19 -0.30 0.01 0.64 0.74 -0.47 -0.11 -0.25 -0.13 0.08 -
0.40 -0.30 -0.51 0.44 058 -0.21 0.00 -0.16
2238 0.06 -0.05 0.01 -0.10 -003 0.11 -0.30 -0.23 0.11 0.12 -0.15 0.39 -0.20
0.01 0.27 0.00 -0.10 016 0.25 -0.41 -0.08
2239 -0.02 0.05 0.09 0.04 046 0.37 -0.49 -0.43 0.52 0.23 -0.15 0.38 -0.17 D.02
0.55 0.01 -0.21 003 0.31 -0.04 0.09
2246 -0.22 0.32 -0.10 -0.04 024 0.18 -0.26 -0.16 0.29 0.08 0.46 -0.17 -0.06
0.36 0.09 0.39 -0.35 -028 -0.18 0.12 0.47
2253 -0.02 0.16 -0.13 0.12 -0.32 -0.12 0.63 0.73 -0.50 -0.11 -0.13 0.05 0.00 -
0.28 -0.36 -0.32 0.36 047 -0.50 -0.05 -0.06
2254 -0.26 -0.03 -0.43 -0.16 -0.22 -0.38 0.40 0.60 -0.38 -0.36 -0.17 0.17 -
0.08 -0.24 -0.47 -0.25 0.17 038 -0.37 0.07 -0.15
2263 -0.07 -0.53 -0.13 -0.17 -0.10 0.06 -0.16 0.1D -0.04 -0.01 -0.57 0.21 -
0.32 -0.08 0.31 -0.25 0.05 043 0.53 -0.28 -0.45
2279 0.17 0.03 0.17 -0.12 034 0.15 -0.48 -0.57 0.37 0.21 0.17 -0.27 -0.03 0.28
0.08 0.29 -0.31 -0.72 0.30 0.39 0.04
2280 0.30 -0.05 0.15 0.02 025 0.09 -0.25 -0.27 0.15 0.26 0.08 -0.46 0.08 0.48
0.06 0.33 -0.27 -059 0.12 0.57 -0.04
2295 -0.10 D.04 0.11 0.21 006 0.26 0.41 0.51 -0.16 -0.05 -0.13 0.00 -0.13 -
D.27 0D7 -0.23 0.36 053 -0.07 -0.06 0.01
2321 -023 -8.08 0 05 0 08 002 -0 05 -026 0.06 0.05 0.12 -021 067 -0.13 -0.23
007 -0.15 -0 33 015 0.11 -015 -0.13
2367 -0.34 -0.63 -0.68 -0.64 -092 -0.77 0.58 0.25 -0.73 -0.63 -0.52 -0.20 0.03
-0.59 -0.70 -0.72 0.73 042 -0.03 -0.63 -0.71
2368 -0.29 -D.42 -0.28 -0.22 -0.41 -0.37 0.38 0.23 -0.29 -0.35 -0.19 -0.20
0.21 -D.35 -0.45 -0.43 0.42 052 0.15 -0.17 -0.33
2383 -0.26 -0.29 -0.28 -0.05 -029 -0.14 0.56 0.76 -0.54 -0.45 -0.22 -0.05 -
0.14 -0.16 -0.34 -0.35 0.47 057 -0.03 -0.09 -0.32
2384 0.06 -0.21 0.07 0.15 -038 -0.09 0.30 0.48 -0.33 0.13 -0.31 0.51 -0.15 -
0.30 -0.02 -0.35 0.16 060 -0.12 -0.36 -0.26
2390 -0.26 -0.31 -0.41 -0.19 -045 -0.40 0.55 0.56 -0.52 -0.49 -0.32 -0.02 -
0.11 -0.44 -0.31 -0.34 0.47 058 -0.24 -0.36 -0.31
2400 -0.32 -0.16 0.18 0.04 0.13 0.23 -0.41 -0.23 0.27 0.23 -0.30 0.20 -0.09 -
0.33 0.26 -0.35 -0.26 007 0.54 0.00 -0.16
2408 -0.45 -0.43 -0.45 -0.39 -0.51 -0.34 0.24 0.22 -0.49 -0.52 -0.41 0.03 -
0.16 -0.41 -0.31 -0.52 0.43 049 0.19 -0.51 -0.50
2425 0.22 -0.26 0.30 0.03 028 0.28 -0.59 -0.37 0.46 0.41 -0.11 0.33 -0.13 0.23
0.37 0.13 -0.40 001 0.69 0.08 -0.13
2441 -0.40 -0.68 -0.71 -0.80 -072 -0.61 0.24 0.06 -0.56 -0.64 -0.63 -0.12 -
0.35 -0.56 -0.43 -0.63 0.56 035 0.22 -0.60 -0.75
2447 0.06 0.68 0.23 0.32 054 0.33 -0.16 -0.29 0.47 0.15 0.66 0.02 0.22 0.36
0.28 0.56 -0.27 -034 -0.37 0.16 0.78
2448 -0.02 0.62 0.40 0.54 016 0.14 0.25 -0.05 0.06 -0.10 0.66 -0.33 0.53 -0.04
-0.D9 0.14 0.06 -029 -0.41 0.32 0.61
2482 0.38 0.16 0.59 0.27 056 0.47 -0.71 -0.36 0.54 0.57 0.32 0.15 -0.22 0.53
0.45 0.58 -0.75 -054 0.41 0.55 0.24
2512 -0.11 D.43 0.02 0.10 019 0.29 0.23 0.28 -0.17 -0.06 0.15 -0.24 -0.09 -
0.03 -0.22 -0.03 0.19 -002 -0.26 0.2D 0.15
2513 0.00 -0.05 0.00 -0.03 024 0.16 0.00 -0.16 0.16 -0.02 0.12 -0.17 -0.06
0.02 -0.01 0.05 0.19 005 0.04 0.13 0.01
2521 0.29 0.29 0.17 0.33 030 0.17 -0.22 0.00 0.28 0.40 0.10 0.45 -0.12 0.39
0.54 0.40 -0.44 004 -0.37 0.15 0.36
2522 -0.08 0.59 0.24 0.44 017 0.07 -0.02 -0.28 0.25 -0.11 0.47 -0.04 0.46 -
0.14 0.16 0.08 -0.12 -030 -0.36 0.18 0.62
2528 -0.25 -0.04 -0.41 -0.16 -012 -0.29 0.38 0.52 -0.31 -0.36 -0.12 0.09 -0.10
-0.22 -0.46 -0.23 0.24 038 -0.34 0.11 -0.14
2529 0.04 0.13 0.30 0.25 002 0.45 0.32 0.49 -0.39 0.06 0.11 -0.26 -0.10 0.26 -
0.16 -0.08 0.29 012 0.15 0.12 -0.08
2544 -0.26 -0.02 -0.43 -0.15 -0.22 -0.37 0.40 0.61 -0.38 -0.36 -0.17 0.16 -
0.08 -0.23 -0.47 -0.25 0.17 038 -0.37 0.07 -0.15
2570 -0.02 -0.19 0.01 -0.17 -0.11 -0.28 0.00 -0.25 0.02 -0.10 0.11 -0.21 0.27 -
0.15 -0.41 -0.06 0.04 -040 0.15 0.09 -0.15
2571 -0.40 0.00 -0.14 -0.24 -0.07 -0.08 -0.32 -0.41 0.16 -0.08 0.12 -0.08 0.06
-0.14 -0.13 -0.11 -0.21 -045 0.18 -0.13 0.04
2586 0.16 D.26 -0.07 0.00 029 0.30 -0.07 0.06 0.14 0.17 -0.14 0.33 -0.09 0.25
0.26 0.13 -0.01 009 -0.05 -0.2D 0.12
2587 -0.25 -0.46 -0.39 -0.39 -053 -0.44 0.26 0.19 -0.34 -0.40 -0.31 0.10 -0.10
-0.45 -0.35 -0.40 0.38 055 0.13 -0.49 -0.42
2603 -0.24 0.07 0.09 0.13 001 0.15 -0.32 -0.10 0.09 0.13 -0.20 0.57 -0.07 -
0.24 0.21 -0.25 -0.27 004 0.19 -0.28 -0.05
2644 0.07 0.09 0.01 -0.21 016 -0.07 -0.45 -0.56 0.28 0.06 0.17 -0.12 -0.03
0.15 0.02 0.26 -0.35 -076 0.10 0.20 0.09
2645 -0.09 -0.20 -0.31 -0.39 -0.19 -0.36 -0.14 -0.35 -0.07 -0.14 -0.22 -0.21
0.01 -0.07 -0.13 -0.09 -0.02 -044 0.01 -0.05 -0.25
2660 -0.22 -0.21 -0.15 0.00 -0.22 0.02 0.30 0.34 -0.29 -0.36 -0.21 -0.17 -0.06
-0.23 0.06 -0.24 0.36 029 0.10 -0.3D -0.16
2683 0.20 0.37 0.28 0.27 055 0.40 -0.58 -0.39 0.46 0.35 0.17 0.28 -0.22 0.48
0.69 0.47 -0.56 -0.34 -0.02 0.28 0.39
2714 0.47 -0.02 0.16 0.18 023 0.07 -0.29 -0.16 0.12 0.35 -0.21 -0.17 -0.06
0.48 0.49 0.29 -0.37 -028 -0.01 0.57 -0.04
2732 -0.44 -0.36 -0.72 -0.70 -073 -0.72 0.43 0.12 -0.64 -0.70 -0.24 -0.32 -
0.11 -0.45 -0.71 -0.42 0.54 003 -0.22 -0.49 -0.46
2733 0.20 -D.14 0.03 0.09 -0.10 -0.05 0.03 -0.22 0.06 0.13 -0.24 -0.25 0.33
D.11 0.31 -0.10 0.11 015 0.08 0.01 -0.05
2807 -0.53 -0.43 -0.65 -0.73 -0.59 -0.57 0.03 -0.17 -0.36 -0.60 -0.33 -0.31 -
0.19 -0.46 -0.45 -0.48 0.28 -001 0.18 -0.35 -0.46
2878 0.41 0.09 0.22 0.37 -0.24 0.03 0.39 0.37 -0.28 0.29 -0.18 -0.07 0.15 D.01
0.13 -0.12 0.15 024 -0.35 0.08 -0.02
2879 -0.34 D.09 -0.18 -0.06 003 0.02 0.24 0.44 -0.20 -0.16 -0.16 -0.11 -0.13 -
0.29 -0.33 -0.31 0.14 026 -0.11 0.26 -0.10
2880 -0.22 -0.29 -0.45 -0.54 -041 -0.48 0.09 -0.16 -0.29 -0.36 -0.21 -0.17 -
0.06 -0.23 -0.40 -0.24 0.28 018 0.04 -0.3D -0.36
2886 0.25 0.47 0.10 0.13 058 0.22 -0.54 -0.40 0.54 0.35 0.33 0.04 -0.16 0.62
0.59 0.76 -0.67 -050 -0.31 0.41 0.58
2936 -0.18 -0.21 -0.35 -0.36 -042 -0.48 0.15 -0.17 -0.27 -0.31 -0.14 -0.23
0.23 -0.22 -0.41 -0.26 0.23 -031 -0.07 -0.26 -0.26
2953 -0.41 -0.44 -0.67 -0.47 -055 -0.61 0.44 0.42 -0.53 -0.66 -0.39 -0.03 -
0.11 -0.42 -0.49 -0.44 0.46 055 -0.11 -0.33 -0.45
3024 -0.12 0.26 0.02 0.28 007 -0.05 0.04 -0.11 0.24 0.07 0.45 -0.19 0.62 0.23 -
0.D4 0.19 -0.20 -018 -0.30 0.15 0.48
3025 -0.06 -0.10 -0.14 0.21 -025 -0.43 0.41 0.26 -0.13 -0.18 -0.01 0.06 0.64 -
0.16 -0.31 -0.23 0.14 025 -0.30 0.07 0.01
3098 -0.17 -0.31 -0.42 -0.49 -050 -0.49 0.16 -0.08 -0.35 -0.33 -0.24 -0.15 -
0.03 -0.27 -0.40 -0.29 0.27 -019 -0.06 -0.35 -0.37
3099 -0.40 -0.28 -0.66 -0.52 -059 -0.69 0.44 0.31 -0.53 -0.69 -0.25 -0.06 -
0.14 -0.50 -0.58 -0.42 0.46 049 -0.28 -0.3D -0.34
3170 -0.22 -0.18 0.15 -0.02 015 0.20 -0.24 -0.16 0.23 0.18 -0.21 -0.17 -0.06 -
0.23 0.14 -0.24 -0.11 005 0.47 0.21 -0.13
3171 -0.12 0.51 0.02 0.13 007 0.23 0.26 0.41 -0.29 -0.05 0.09 -0.17 -0.06 -
0.04 -0.24 -0.06 0.10 -0.06 -0.32 0.15 0.16
3172 -0.29 -0.36 -0.60 -0.60 -0.49 -0.63 0.20 0.03 -0.38 -0.48 -0.28 -0.07 -
0.08 -0.30 -0.53 -0.32 0.32 032 -0.06 -0.28 -0.42
3390 -0.12 -0.15 -0.27 0.07 -0.27 -0.48 0.40 0.36 -0.20 -0.26 -0.09 0.13 0.43 -
0.19 -0.37 -0.25 0.15 033 -0.31 0.05 -0.07
3463 0.10 -0.27 -0.18 -0.02 -0.50 -0.34 0.53 0.58 -0.41 -0.07 -0.30 0.26 -0.09
-0.33 -0.20 -0.34 0.34 066 -0.32 -0.26 -0.26
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
202
Table 31 (cont.). Correlation matrix for acidic glycans derived from embryonic
stem cells.
2157 2164 2221 2222 2230 2237 2238 2239 2248 2253 2254 2263 2279 2280 2295
2321 2387 2368 2383 2384 2390 2400 2408
1354 0.01 0.07 -0.23 -0.37 0.60 -0.33 -0.14 -0.10 0.59 -0.24 -0.19 -0.43 0.04
D.03 -0.09 -0.02 -0.45 -0.18 -0.27 -0.20 -0.12 -0.21 -0.34
1362 -0.28 0.18 0.37 -0.23 0.10 0.27 -0.21 0.32 -0.09 0.33 0.08 -0.29 0.17
D.19 0.23 -0.19 -0.17 -0.19 0.08 -0.10 -0.01 -0.13 -0.08
1403 -0.20 0.04 0.22 -0.22 0.17 0.54 -0.12 -0.17 -0.06 0.64 0.29 -0.32 -0.04
D.04 0.19 -0.13 -0.10 -0.17 0.03 -0.08 -0.02 -0.09 -0.10
1475 0.00 -0.30 -0.29 -0.39 0.24 -040 0.15 0.07 0.25 -0.36 -0.24 0.17 0.42
D.46 -0.50 0.08 -0.59 -033 -034 -0.18 -0.44 0.19 -0.28
1500 -0.07 0.10 -0.48 -0.37 0.68 -048 0.26 0.26 0.54 -0.38 -0.34 0.03 0.09 -
D.01 -0.13 0.19 -0.75 -047 -043 -0.09 -0.32 0.11 -0.36
1516 -0.07 0.24 -0.15 -0.37 0.41 -0.15 -0.15 -0.26 0.70 -0.09 -0.11 -0.34 0.02
D.22 -0.07 0.05 -0.42 -0.16 -007 -0.10 -0.11 -0.22 -0.25
1541 -0.17 0.13 -0.23 -0.37 0.66 -027 -0.10 -0.04 0.46 -0.13 -0.12 -0.43 0.05 -
D.05 0.04 0.00 -0.47 -0.29 -023 -0.17 -0.10 -0.20 -0.32
1549 -0.24 -0.16 -0.16 0.40 -0.22 023 0.05 -0.21 -0.08 0.03 -0.01 0.53 -0.33 -
D.14 0.32 -0.16 0.10 0.11 045 -0.17 0.72 -0.11 0.43
1557 0.05 -0.22 0.06 -0.33 0.43 010 -0.17 -0.05 -0.11 0.14 0.00 -0.56 -0.01 -
D.17 0.07 -0.23 -0.16 -005 -017 -0.24 -0.21 -0.16 -0.26
1565 -0.16 0.34 -0.18 -0.13 0.10 0.15 -0.20 0.03 0.20 0.20 0.04 -0.11 -0.30
0.02 0.37 -0.33 -0.37 -020 0.27 -0.02 0.16 -0.52 -0.24
1637 0.02 -0.17 -0.31 -0.41 0.35 -0.45 0.20 0.16 0.28 -0.41 -0.27 0.17 0.41
0.37 -0.42 0.01 -0.63 -033 -0.39 -0.25 -0.48 0.16 -0.36
1678 0.12 0.06 -0.44 -0.52 0.38 -029 -0.02 0.21 0.13 -0.34 -0.28 0.02 0.23
D.32 -0.10 0.06 -0.82 -030 -023 -0.15 -0.42 0.09 -0.48
1703 0.11 0.15 -0.33 -0.47 0.71 -035 0.22 0.36 0.18 -0.15 -0.17 -0.22 0.05
0.00 -0.19 0.36 -0.70 -060 -037 -0.02 -0.39 0.04 -0.24
1711 0.00 -0.19 0.13 -0.06 -0.21 -0.44 0.24 0.34 -0.09 -0.46 -0.37 0.32 0.50
D.42 -0.42 -0.17 0.09 -020 -013 -0.30 -0.36 0.09 0.15
1719 0.09 0.16 -0.26 -0.42 0.85 -026 0.18 0.35 0.32 -0.11 -0.17 -0.38 -0.07 -
D.26 -0.08 0.30 -0.57 -038 -032 -0.03 -0.37 0.04 -0.21
1727 -0.06 0.00 -0.21 -0.31 -0.12 026 -0.38 -0.03 -0.04 0.02 -0.04 0.04 0.07
D.35 0.33 -0.34 -0.49 002 010 -0.28 -0.04 0.00 -0.43
1744 -0.06 0.28 -0.20 -0.37 0.46 -0.26 -0.05 -0.31 0.35 -0.16 -0.14 -0.21 0.19
D.25 -0.12 0.07 -0.46 -0.19 -006 -0.09 -0.14 -0.23 -0.26
1768 D.16 0.43 -0.03 -0.37 0.24 010 -0.31 -0.18 -0.16 0.10 -0.05 -0.48 0.03
D.17 0.23 0.10 -0.27 -009 007 -0.02 -0.02 -0.22 -0.19
1791 -D.14 0.05 -0.22 -0.31 0.58 -030 -0.04 -0.18 0.74 -0.18 -0.12 -0.31 0.00
D.00 -0.12 0.01 -0.43 -022 -024 -0.16 -0.11 -0.17 -0.29
1799 D.10 -0.11 -0.20 -0.38 0.22 -037 0.00 -0.02 -0.15 -0.22 -0.16 -0.06 0.40
D.43 -0.31 -0.15 -0.48 -0.36 -027 -0.26 -0.24 -0.21 -0.35
1840 D.24 0.14 -0.13 -0.26 0.05 -017 0.09 -0.02 -0.22 -0.02 -0.26 -0.07 0.17
D.30 -0.10 -0.23 -0.34 -0.29 -026 0.09 -0.26 -0.32 -0.45
1865 -0.14 0.03 -0.17 -0.55 0.78 -001 -0.05 0.05 0.32 0.16 -0.03 -0.53 0.03 -
0.05 0.04 -0.08 -0.63 -042 -029 -0.21 -0.31 -0.16 -0.43
1873 -0.04 -0.07 -0.35 -0.48 0.38 -003 0.01 0.09 -0.10 -0.13 -0.43 -0.13 0.17
D.15 0.11 0.05 -0.68 -028 -028 0.07 -0.41 0.18 -0.45
1889 -0.01 -0.01 -0.33 -0.39 0.34 019 -0.10 0.04 -0.04 0.12 -0.16 -0.17 -0.12
D.02 0.21 0.08 -0.64 -022 -005 0.15 -0.19 0.04 -0.39
1906 -0.08 0.28 -0.43 -0.63 0.64 -030 -0.03 0.46 0.24 -0.32 -0.22 -0.10 0.34
D.25 0.06 0.02 -0.92 -041 -029 -0.38 -0.45 0.13 -0.51
1914 -0.39 0.04 -0.40 -0.44 0.46 001 0.11 0.37 0.18 -0.12 -0.38 0.06 0.15 D.09
0.26 -0.05 -0.77 -037 -014 -0.09 -0.40 0.23 -0.34
1930 -0.15 0.04 0.33 0.40 -0.46 064 -0.30 -0.49 -0.26 0.63 0.40 -0.16 -0.48 -
D.25 0.41 -0.26 0.58 0.38 056 0.30 0.55 -0.41 0.24
1946 -D.37 0.23 0.12 0.34 -0.36 074 -0.23 -0.43 -0.16 0.73 0.60 0.10 -0.57 -
D.27 0.51 0.06 0.25 023 0,76 0.48 0.56 -0.23 0.22
1947 D.15 0.05 -0.40 -0.50 0.59 -0.47 0.11 0.52 0.29 -0.50 -0.38 -0.04 0.37
D.15 -0.16 0.05 -0.73 -029 -054 -0.33 -0.52 0.27 -0.49
2002 0.13 0.11 -0.29 -0.43 0.26 -0.11 0.12 0.23 0.08 -0.11 -0.36 -0.01 0.21
0.26 -0.05 0.12 -0.63 -035 -045 0.13 -0.49 0.23 -0.52
2010 -D.12 -0.06 -0.16 -0.54 0.72 -025 -0.15 -0.15 0.46 -0.13 -0.17 -0.57 0.17
0.08 -0.13 -0.21 -0.52 -019 -022 -0.31 -0.32 -0.30 -0.41
2011 D.20 0.42 -0.25 0.10 0.35 -0.13 0.39 0.38 -0.17 0.05 0.17 0.21 -0.27 -
0.46 0.00 0.67 -0.20 -0.20 -005 0.51 -0.02 0.20 0.03
2018 0.48 -0.17 0.10 -0.17 0.02 008 -0.20 -0.17 -0.06 0.00 -0.08 -0.32 -0.03
0.08 -0.13 -0.13 0.03 021 -014 -0.15 -0.11 -0.09 -0.16
2035 0.09 0.22 -0.20 -0.48 0.27 -040 0.01 0.02 0.36 -0.28 -0.24 -0.08 0.28
D.48 -0.27 -0.23 -0.59 -035 -0.16 -0.30 -0.44 -0.33 -0.41
2052 -0.01 0.02 -0.51 -0.25 0.29 -030 0.27 0.55 0.09 -0.36 -0.47 0.31 0.08
0.06 0.07 0.07 -0.70 -045 -034 -0.02 -0.31 0.26 -0.31
2068 D.09 0.23 -0.31 -0.54 0.60 -0.51 0.00 0.01 0.39 -0.32 -0.25 -0.25 0.29
0.33 -0.23 -0.15 -0.72 -043 -035 -0.35 -0.34 -0.35 -0.52
2076 -D.17 -0.04 0.32 0.52 -0.55 044 -0.10 -0.21 -0.35 0.36 0.17 0.05 -0.31 -
0.27 0.36 -0.33 0.73 042 047 0.16 0.47 -0.26 0.43
2092 -0.06 0.23 -0.14 0.73 -0.51 058 0.16 0.03 -0.28 0.47 0.38 0.43 -0.72 -
0.59 0.53 0.15 0.42 052 057 0.6D 0.58 0.07 0.49
2117 -0.02 -0.15 -0.16 0.07 -0.21 -0.21 0.25 0.31 -0.18 -0.50 -0.37 0.53 0.30
D.12 -0.07 0.11 -0.03 015 -003 -0.12 -0.24 0.54 0.19
2133 -0.07 -0.02 -0.14 -0.61 0.17 000 -0.41 -0.04 0.12 -0.05 0.07 -0.28 0.39
D.57 -0.06 -0.15 -0.63 -017 -009 -0.36 -0.36 0.00 -0.51
2156 -0.08 0.00 -0.28 -0.55 0.79 -0.16 -0.08 0.09 0.47 -0.06 -0.15 -0.45 0.04 -
D.04 0.01 -0.13 -0.71 -0.33 -0.32 -0.26 -0.31 -0.16 -0.50
2157 1.00 0.06 -0.28 0.30 -0.01 -032 0.41 -0.09 -0.20 -0.22 -0.05 0.10 -0.23 -
D.15 -0.41 0.12 0.12 040 -025 -0.15 0.05 -0.12 0.33
2164 0.06 1.00 -0.10 -0.09 0.18 006 -0.23 0.18 -0.17 0.15 0.45 -0.14 -0.20 -
0.17 0.46 0.30 -0.21 -002 041 0.12 0.17 -0.24 -0.10
2221 -0.28 -0.10 1.00 -0.34 -0.32 0.21 -0.61 -0.28 -0.09 0.29 0.20 -0.54 0.51
D.47 -0.14 -0.24 0.56 -0.25 -0.01 -0.03 -0.21 -0.16 -0.35
2222 D.30 -0.09 -0.34 1.00 -0.50 0.17 0.53 -0.16 -0.17 0.11 0.13 0.67 -0.73 -
0.60 0.12 0.06 0.55 066 032 0.30 0.72 -0.01 0.82
2230 -0.01 0.18 -0.32 -0.50 1.00 -038 0.23 0.30 0.27 -0.18 -0.20 -0.37 0.08 -
0.23 -0.11 0.20 -0.68 -045 -036 -0.12 -0.42 -0.06 -0.31
2237 -0.32 0.06 0.21 0.17 -0.38 1,00 -0.37 -0.22 -0.19 0.85 0.54 -0.06 -0.37 -
D.18 0.62 -0.05 0.19 030 040 0.38 0.35 0.15 -0.06
2238 0.41 -0.23 -0.51 0.53 0.23 -037 1.00 0.24 0.02 -0.24 -0.41 0.52 -0.41 -
D.55 -0.30 0.16 -0.04 0.11 -034 0.15 0.02 0.12 0.55
2239 -0.09 0.18 -0.28 -0.16 0.30 -0.22 0.24 1.00 -0.17 -0.28 -0.22 0.16 0.18 -
0.18 0.18 0.21 -0.38 -0.29 -024 -0.03 -0.36 0.46 -0.08
2246 -0.20 -0.17 -0.D9 -0.17 0.27 -0.19 0.02 -0.17 1.00 -0.13 -0.08 -0.06 -
0.01 0.15 -0.31 -0.13 -0.25 -0.06 -0.14 -0.15 -0.17 -0.09 -0.16
2253 -D.22 0.15 0.29 0.11 -0.18 0,85 -0.24 -0.28 -0.13 1.00 0.60 -0.29 -0.41 -
D.20 0.37 -0.03 0.19 006 028 0.52 0.26 -0.19 -0.12
2254 -0.05 0.45 0.20 0.13 -0.20 054 -0.41 -0.22 -0.08 0.60 1.00 -0.08 -0.35 -
D.14 0.22 0.30 0.15 032 069 0.23 0.36 -0.12 0.11
2263 D.10 -0.14 -0.54 0.67 -0.37 -006 0.52 0.16 -0.06 -0.29 -0.08 1.00 -0.38 -
D.26 0.05 0.20 0.04 035 027 0.05 0.48 0.34 0.64
2279 -D.23 -0.20 0.51 -0.73 0.08 -037 -0.41 0.18 -0.01 -0.41 -0.35 -0.38 1.00
0.78 -0.34 -0.22 -0.11 -051 -043 -0.48 -0.65 0.15 -0.54
2280 -0.15 -0.17 0.47 -0.60 -0.23 -018 -0.55 -0.18 0.15 -0.20 -0.14 -0.26 0.78
1.00 -0.38 -0.29 -0.09 -039 -014 -0.42 -0.35 -0.11 -0.45
2295 -0.41 0.46 -0.14 0.12 -0.11 062 -0.30 0.18 -0.31 0.37 0.22 0.05 -0.34 -
D.38 1.00 -0.05 -0.10 016 040 0.19 0.38 0.14 -0.12
2321 D.12 0.30 -0.24 0.06 0.20 -005 0.16 0.21 -0.13 -0.03 0.30 0.20 -0.22 -
D.29 -0.05 1.00 -0.18 -004 021 0.52 0.06 0.46 0.27
2367 D.12 -0.21 0.56 0.55 -0.68 019 -0.04 -0.38 -0.25 0.19 0.15 0.04 -0.11 -
D.09 -0.10 -0.18 1.00 041 016 0.15 0.34 -0.13 0.42
2368 0.40 -0.02 -0.25 0.66 -0.45 030 0.11 -0.29 -0.06 0.06 0.32 0.35 -0.51 -
D.39 0.16 -0.04 0.41 1,00 038 -0.05 0.45 0.12 0.56
2383 -0.25 0.41 -0.01 0.32 -0.36 040 -0.34 -0.24 -0.14 0.28 0.69 0.27 -0.43 -
0.14 0.40 0.21 0.16 038 1,00 0.17 0.63 -0.19 0.40
2384 -D.15 0.12 -0.03 0.30 -0.12 038 0.15 -0.03 -0.15 0.52 0.23 0.05 -0.48 -
D.42 0.19 0.52 0.15 -0.05 017 1.00 0.18 0.11 0.08
2390 0.05 0.17 -0.21 0.72 -0.42 035 0.02 -0.36 -0.17 0.26 0.36 0.48 -0.65 -
0.35 0.38 0.06 0.34 045 063 0.18 1.00 -0.24 0.58
2400 -0.12 -0.24 -0.16 -0.01 -0.06 015 0.12 0.46 -0.09 -0.19 -0.12 0.34 0.15 -
D.11 0.14 0.46 -0.13 012 -019 0.11 -0.24 1.00 0.05
2408 0.33 -0.10 -0.35 0.82 -0.31 -0.06 0.55 -0.08 -0.16 -0.12 0.11 0.64 -0.54 -
D.45 -0.12 0.27 0.42 056 040 0.08 0.58 0.05 1.00
2425 0.35 -0.18 -0.64 0.24 0.09 -035 0.63 0.29 0.03 -0.47 -0.25 0.59 -0.06 -
D.18 -0.29 0.29 -0.29 031 -019 -0.01 -0.20 0.48 0.31
2441 0.02 -0.29 0.28 0.64 -0.62 -0.07 0.22 -0.07 -0.27 -0.13 -0.03 0.43 -0.03 -
D.13 -0.12 -0.11 0.82 0.30 012 0.0D 0.37 0.07 0.59
2447 0.02 0.40 -0.21 -0.44 0.72 -0.31 -0.06 0.25 0.31 -0.20 -0.20 -0.49 0.00 -
D.17 0.19 -0.17 -0.50 -0.26 -023 -0.29 -0.31 -0.30 -0.41
2448 0.05 0.01 -0.01 -0.41 0.45 002 -0.26 -0.05 -0.16 0.03 -0.08 -0.63 0.03 -
0.09 0.16 -0.15 -0.25 -0.07 -007 -0.19 -0.22 -0.23 -0.27
2482 -0.04 -0.18 -0.35 -0.39 0.35 -045 0.17 0.01 0.23 -0.46 -0.29 0.14 0.36
0.31 -0.39 0.26 -0.61 -028 -027 -0.08 -0.46 0.27 -0.24
2512 -0.28 0.13 0.17 -0.26 0.16 047 -0.19 0.09 -0.09 0.56 0.21 -0.35 0.08 D.14
0.24 -0.19 -0.15 -021 006 -0.10 -0.02 -0.13 -0.10
2513 -0.20 0.17 -0.05 -0.13 0.02 -0.02 -0.17 0.46 -0.06 -0.01 -0.08 -0.13 0.22
D.20 0.15 -0.13 -0.13 -011 007 -0.06 0.00 -0.09 -0.03
2521 -0.02 0.36 -0.29 -0.16 0.31 -0.16 0.10 0.25 0.28 0.09 0.09 -0.10 -0.26 -
D.15 0.01 0.31 -0.46 -0.40 -006 0.46 -0.17 -0.15 -0.30
2522 0.12 -0.20 -0.10 -0.31 0.53 -0.16 0.03 0.29 -0.15 -0.09 -0.13 -0.40 0.02 -
D.25 -0.04 -0.03 -0.28 -0.19 -027 -0.15 -0.32 -0.02 -0.20
2528 -0.12 0.49 0.18 0.07 -0.18 052 -0.45 -0.04 -0.10 0.58 0.93 -0.13 -0.26 -
D.06 0.27 0.24 0.09 026 068 0.2D 0.35 -0.15 0.09
2529 -0.31 0.24 0.09 -0.21 0.04 024 -0.14 -0.18 -0.10 0.23 0.07 -0.23 0.00
0.09 0.23 -0.01 -0.10 -0.06 035 0.07 -0.16 -0.14 0.03
2544 -0.05 0.44 0.20 0.12 -0.19 055 -0.41 -0.22 -0.08 0.61 1.00 -0.08 -0.35 -
D.14 0.22 0.30 0.14 032 068 0.23 0.36 -0.12 0.11
2570 -0.07 -0.05 0.59 -0.47 -0.12 -007 -0.63 -0.21 -0.22 -0.18 -0.02 -0.49
0.70 0.49 -0.12 -0.07 0.30 -0.08 -010 -0.25 -0.28 0.08 -0.39
2571 -0.26 -0.45 0.53 -0.40 0.11 -025 -0.18 0.04 0.41 -0.30 -0.22 -0.23 0.59
0.35 -0.47 0.11 0.16 -0.29 -0.35 -0.15 -0.49 0.38 -0.22
2586 0.02 0.47 0.D3 -0.12 0.29 009 0.15 0.39 -0.09 0.20 0.07 -0.07 -0.09 -0.24
0.31 -0.19 -0.18 -0.22 -0.11 -0.18 -0.16 -0.12 -0.19
2587 0.41 -0.17 -0.41 0.92 -0.34 006 0.55 -0.25 -0.09 0.01 0.11 0.60 -0.65 -
0.59 -0.04 0.08 0.47 0,76 023 0.18 0.62 0.02 0.80
2603 0.05 -0.16 -0.19 0.03 0.32 -0.11 0.47 0.40 -0.07 -0.03 -0.04 0.22 -0.10 -
0.28 -0.28 0.75 -0.17 -027 -013 0.40 -0.17 0.51 0.30
2644 -0.17 -0.27 0.60 -0.66 0.16 -047 -0.33 0.05 0.05 -0.41 -0.31 -0.41 0.90
0.63 -0.47 -0.14 0.02 -058 -047 -0.39 -0.60 0.05 -0.51
2645 -0.13 -0.18 0.79 -0.39 -0.25 -026 -0.43 0.00 -0.07 -0.23 -0.17 -0.32 0.74
0.63 -0.33 -0.15 0.39 -044 -029 -0.27 -0.33 0.02 -0.32
2660 -0.20 -0.17 -0.22 0.46 -0.27 0.10 0.08 -0.17 -0.06 -0.13 -0.08 0.62 -0.33
-D.15 0.28 -0.13 0.12 015 045 -0.15 0.73 -0.09 0.46
2683 -0.01 0.16 -0.40 -0.33 0.50 -0.58 0.28 0.57 0.11 -0.42 -0.28 0.02 0.13
D.02 -0.18 0.28 -0.62 -057 -024 0.00 -0.43 0.04 -0.15
2714 0.14 -0.17 -0.08 -0.17 -0.27 -0.19 -0.03 0.04 -0.06 -0.13 -0.08 0.17 0.24
D.55 -0.31 -0.13 -0.25 -0.30 -014 -0.15 -0.17 -0.09 -0.16
2732 -0 .08 -0.14 0.70 0.22 -0.44 -004 -0.24 -0.43 -0.04 0.04 0.08 -0.18 0.17
D.17 -0.21 -0.19 0.84 006 0.12 -0.06 0.26 -0.30 0.24
2733 0.48 -0.24 -0.25 0.33 -0.31 -0.05 0.33 0.10 -0.08 -0.17 -0.32 0.24 -0.21 -
D.15 0.02 -0.44 0.14 0.38 -031 -0.23 -0.07 0.04 0.11
2807 0.07 -0.50 0.30 0.43 -0.46 -017 0.19 -0.20 0.08 -0.25 -0.07 0.30 0.14
0.05 -0.44 -0.15 0.68 033 -008 -0.24 0.11 0.19 0.48
2878 -0.07 -0.24 0.17 0.04 -0.27 053 -0.02 -0.24 -0.12 0.68 0.00 -0.18 -0.22
0.04 0.12 -0.26 0.10 -019 -020 0.49 0.01 -0.18 -0.29
2879 -0.28 0.25 0.20 -0.08 -0.20 0,76 -0.46 0.03 -0.13 0.59 0.70 -0.10 -0.06
0.01 0.47 0.11 -0.01 028 039 0.01 0.13 0.32 -0.07
2880 0.61 -0.17 -0.21 0.65 -0.27 -0.19 0.55 -0.17 -0.06 -0.13 -0.08 0.25 -0.29
-0.29 -0.31 -0.13 0.50 0.60 -014 -0.15 0.20 -0.09 0.68
2886 0.05 0.16 -0.28 -0.34 0.39 -0.49 0.07 0.15 0.51 -0.33 -0.20 -0.07 0.12
D.20 -0.19 -0.13 -0.55 -042 -034 -0.28 -0.28 -0.22 -0.39
2936 -0.07 -0.21 0.89 -0.29 -0.24 -0.12 -0.44 -0.23 -0.08 -0.08 -0.08 -0.43
0.58 D.47 -0.33 -0.18 0.58 -027 -017 -0.20 -0.20 -0.12 -0.21
2953 0.27 0.12 -0.15 0.79 -0.49 020 0.11 -0.31 -0.11 0.13 0.57 0.51 -0.60 -
0.36 0.07 0.10 0.50 0,72 066 0.02 0.79 -0.16 0.76
3024 0.18 -0.13 0.04 -0.19 0.15 000 -0.21 -0.28 0.71 0.00 0.12 -0.26 -0.12
D.12 -0.28 -0.10 -0.13 020 -002 -0.15 -0.11 -0.15 -0.19
3025 0.35 0.23 0.17 0.05 -0.19 0.34 -0.43 -0.24 -0.09 0.30 0.66 -0.19 -0.29 -
D.07 0.04 0.19 0.16 0.45 045 0.10 0.22 -0.13 0.01
3098 -0.22 -0.21 0.89 -0.18 -0.31 -0.09 -0.37 -0.21 -0.08 -0.01 -0.09 -0.35
0.53 D.40 -0.26 -0.17 0.64 -032 -017 -0.01 -0.15 -0.11 -0.20
3099 0.35 0.04 -0.14 0.77 -0.37 016 0.23 -0.29 -0.14 0.21 0.50 0.27 -0.60 -
D.50 0.01 0.03 0.55 0,72 041 0.10 0.58 -0.20 0.68
3170 -0.20 -0.17 -0.04 -0.05 -0.27 031 -0.19 0.24 -0.06 -0.13 -0.08 0.21 0.24
D.06 0.38 0.02 -0.05 0.31 -0.14 -0.15 -0.17 0.82 -0.16
3171 -0.20 0.04 0.22 -0.22 0.17 0.54 -0.12 -0.17 -0.06 0.64 0.29 -0.32 -0.04
D.04 0.19 -0.13 -0.10 -017 003 -0.08 -0.02 -0.09 -0.10
3172 0.59 0.01 -0.15 0.70 -0.35 -0.05 0.39 -0.22 -0.08 0.02 0.27 0.25 -0.41 -
D.33 -0.24 0.01 0.55 0,72 013 -0.04 0.34 -0.12 0.70
3390 0.25 0.32 0.17 0.11 -0.23 037 -0.44 -0.23 -0.09 0.36 0.81 -0.12 -0.34 -
D.11 0.08 0.26 0.18 046 057 0.17 0.30 -0.12 0.06
3463 -0.16 0.10 0.15 0.38 -0.38 057 -0.11 -0.24 -0.09 0.69 0.44 -0.02 -0.50 -
D.34 0.30 0.08 0.35 018 027 0.80 0.34 -0.13 -0.05
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
203
Table 31 (cont.). Correlation matrix for acidic glycans derived from embryonic
stem cells.
11 1354 ?9.18 29.53 om Z9.4fi 9.19 9.99 2111 2121 Z9a4 -91s 2 az9 ?9.19 2171
29.1s 2az1 2az4 29.n 29.11 19.99 19.1s 2111 29.1s 29nz ?9.14 ?aa 1az1 1111
29.1s 29s] 29.19 2111 112, 1121 1111 39.n -9.1s 19.is 39.19 -0.94 1.21
136 -9.39 -9.18 9.99 9.17 -9.31 U.6 U85 9.49 Q29 9.99 9.95 -9.15 1s ~9z] -9.91
3 -0.98 ~9.99 -9.96 -9.99 -9.9] -9.31 -9.39 9.97 9.42 -9.99 -9.22 9.97 -9.16 -
0.15 -9.13 -9.19 -Q1fi -9.99 9.4] -9.12 -0.12 d.13
1403 -9.38 -Q23 9.91 Q20 -9.22 0.86 -99fi -9.11 9.03 9.z] 94] 9.31 11 1 9.44
~Q24 9.99 9 -9.98 ~99fi -9.22 -99fi 9.92 -9.19 -91fi 9.34 9s8 -9.96 -91fi 9.99
-9.11 -0.11 9.99 -9.95 -Q98 -9.9fi 1.00 -9.98 -0.99 d09
14]5 9.58 -9.33 -9.14 -Q21 0.91 9.17 -9.19 9.29 -9.05 -9.27 -0.15 -9.24 ~9.96
9.15 -9.25 ~9.33 9.16 9.31 -9.92 9.29 9.42 95B 9.49 -9.98 -9.95 -9.92 9.19 -
9z0 9.44 927 -93] 0.91 .29 -9.25 -9.38 9.99 9.14 -926 -9.29 d28 52 -9.15 -9.59
9.52 9.18 9.z3 9.13 9.99 9.58 -9.16 -9.9] 9.26 -9.11 9.95 9.14 -9.99 9.38 -
9.14 -9.95 -0.15 :9.15 9.12 -9.15 9.11 -9.27 -9.39 -9.14 .9.19 -9.15 9.42
~9.12 -9.28 8.55 d.9fi -9.18 -9.32 -9.15 9.99 -Q29 -0.19 d.22
56 -9.z4 -9.43 9.fi] 9.45 9.21 9.98 9] 9.33 9.35 -9.99 -9.99 -9.1z ~99fi 9fi -
9.19 9.35 -9.13 9.13 -9.11 9.14 9.35 -9.14 9.99 -9.zfi -9.25 -9.z9 9.1z -0.14
9.56 ~9.1] -926 0.22 1.29 -9.1] -Q10 -9.14 9.95 -9.9 -0.9 1.29
154 -9.19 Qz] -9.21 -9.11 -9.z] 9.13 -9.98 -9.3fi -9.14 -9.93 9.92 -9.91 ~9.25
-9.22 9.92 Q29 5 -9.31 -91] 09] -927 -99B 9.14 -9.95 0.14 -0.91 9.92 -9.98 -
9.19 ~9.98 042 -0.13 111 -9.99 Q12 99B 9.19 -9.9 -0.11 11
155] -9.35 -9.34 9.49 0.96 -9.1fi 9.2 1 -9.11 -9.96 U.89 -9.94 9.95 9.91 9.11 -
9.91 95 9.35 -9.9fi 9.19 -0.9] 9.11 99] -9.11 9.11 9.16 -9.15 9.12 9.94 -9.11
9.93 9.97 -9.z9 0.24 9.27 -9.19 9.91 -9.11 9.39 -9.15 0.15 d1fi
1565 -9.49 -9.48 9.53 Q29 -9.25 9.29 9.18 9.59 91fi 9.11 925 9.94 ~9.44 -9.48
9.35 ~9.33 -9.35 -936 -9z] 9.23 9.28 9.24 9.39 9.21 -9.63 9.31 9.98 -9.36 9.43
9.28 -9.99 0.39 9.15 -9.33 Q19 -93fi 9.11 -9.35 0- 9.10
13] 9.61 -9.3fi 9.91 -9.25 0.88 ~9.z1 -9.13 9.21 -9.06 -9.39 -9.2 -927 ~9fi
912 -9.93 ~9.33 9.99 936 -0.95 22 9.42 946 9.54 -9.94 -9.1z -9.11 ~9.21 -9.zz
9.51 ~9.z9 -9.49 0.91 d.3z -9.28 9.2 9.11 9.16 -929 -9.39 d.31
i]8 9.42 -05 9.31 917 ]0 ~9.18 -9.91 9.39 9.19 -9.28 9.91 -9.29 9.99 -9.13
9.99 9.43 9.92 9.19 -9.22 9.15 9.59 9.46 U.]9 9.11 -9fi9 9.93 ~9.23 -4] 9.49
~9.49 -952 0.23 9.91 -9.59 Q61 .9B .29 -9.53 -0.9] 2
1]03 9.92 -9.58 9.53 9.32 9.34 9.15 -997 69 9.49 -9.19 9.93 -91] 9.34 9.92 925
9.49 9.48 9.13 -9.95 9.26 0.80 9.31 9.46 -9.9fi -9.44 9.92 9.18 -QZfi 9.63
9.23 -04] 0.99 d.18 -9.39 Q40 -92fi 921 -9.34 -Q22 d3fi
1]11 93] 9.34 -9.31 -9.43 9.21 9.93 9.23 -9.24 -9.28 -9z] 9.13 -93] 9] 9.24
9.14 9.19 9.14 93fi 9.35 9.91 9.29 9.32 9.9] 9.19 9.39 -9.22 9.2fi 9.12 -99fi
92z -9.15 -0z9 d.39 9.23 Q27 -9.91 9.16 9.99 -0.39 d.42
1719 -9.9fi -9.fiz U.]4 9.51 9.98 9.16 992 9.41 9.69 9.15 9.95 -9.1] ~9.29
9.19 9.29 9.37 9.45 9.93 -9.18 9.24 952 -9.24 9.38 -9.13 9.4 -9.16 9.1fi -9.z4
9.31 9.14 -0.44 0.39 9.91 -9.27 -Q36 -9.24 9.17 -9.32 -0.99 d.34
]3] 9.91 -9.53 9.14 Qzfi z1 9.94 9.99 9.19 9.13 -9.93 9.11 -9.94 ~99fi -936
9.94 9.35 -9.42 -9.13 -9.19 1fi 9.97 1.4 ' 9.57 9.36 9.7 9.29 9.16 -9.43 9.2fi
9.31 -921 0.21 9.22 -9.42 Q35 9.29 9.95 -9.43 0.13 d.08
1]44 9.16 -9.51 926 9.93 57 9.98 9.95 9.95 -9.33 -9.11 9.3 -9.14 9.14 -9.91 -
9.12 ~9.1fi -9.15 9.99 -9z3 1fi 9.93 -9.16 9.31 -9.49 9.1 -9.Zi 9.14 -9.1fi
9.21 9.z9 -9.39 0.12 d.24 -9.29 Q35 -9.1fi 9.96 -922 -0.23 d.23
18 -9.39 -9.57 9.38 56 -9.95 9.38 z5 -9.91 07 9.95 9.53 -9.95 9.21 -9z4 9.99
~9.34 -9.11 -9.14 -9.13 ~~9.1fi -9.93 -9.1fi ~9.14 -9.29 964 9.92 9.96 -9.1fi -
9.99 9.91 -9.29 0.12 9.1fi -9.16 Q32 -9.1fi 9.28 -9.21 0.95 22
1]6]91 -9.13 -9.43 9.69 Q23 9.30 ~9.98 -9.12 9.38 9.15 -9.16 -9.13 -9.12 9.12
9.21 2 9.19 -9.12 9.12 -0.10 9.12 9.29 -9.12 ~9] 29 -9.14 -0.19 9.18 -9.12
9fifi 9.15 -9.22 0.45 d.1] -9.15 Q12 -9.12 9.92 -9.16 -0.16 17
1]9 9.25 -9.27 -9.92 9.98 965 ~9.93 9.94 9.12 9.13 -9.14 -9.16 -91fi 9.95 -9z3
fi 24 -91fi 936 9.2 ~9.15 3fi fi1 9.34 -9.95 -9.19 9.98 -0.29 -9.15 9.49 9.19 -
927 -0.25 d.2z -9.18 Q21 -9.15 9.96 -929 -0.21 d21
184 9.22 -9.49 9.96 -9.92 9.38 9.11 9.99 9.z9 -9.08 -9.z5 9.94 -9zfi 9.92 -
9.49 9.16 9.35 -9.24 9.9] -9.99 ~2z 29 41 9.44 29 -9.53 9.41 9.34 -9.22 9.25
9.18 -9.41 -0.12 d.9fi -9.17 -Q40 -9.22 9.12 -929 -0.12
9.10
186 -9.zfi -9fi8 9fi8 9fi2 91fi 9.43 -9.95 9z9 9.59 -9.94 9.13 -9.9 9.19 9.99
9.26 9.46 9.97 9.99 -9.z9 ~9.z1 937 -9.92 ~9.3fi -9.14 -9.43 9.99 9.99 -9.z9
9.47 ~9.z1 -044 0.26 d.19 -9.31 -9.28 -9.1B 9.51 -9.36 -0.15 27
18]3 9.39 -0I1 9.23 9.40 9.59 9.92 9.99 9.17 9.24 -9.41 9.39 -9.43 9.91 -9.14 -
9.9] ~9.39 9.99 9.91 -0.31 ~9.15 9.28 9.1fi ~0]2 9.93 -9fi5 9.22 9.18 -9.45
9.19 ~9.35 -9.fi] 0.92 d.14 -9.42 -966 9.15 9.92 -9.69 -QZ] d18
1889 9.93 -0.80 9.32 9.54 9.z] 9.19 -9.93 9.33 9.44 -91fi Q25 -9.15 91] -9z4
9.99 ~9.39 9.13 -Q21 -0.39 9.99 927 9.18 0.70 9.99 -0]3 937 9.96 -9.54 9.13
9.36 -047 Q28 9.21 -9.49 -9.52 -9.92 9.13 -.fi9 0.9] d92
1906 928 -0I2 9.54 916 956 9.19 9.24 9.39 9.17 -9.12 9.92 -9.22 9.11 -99] 9.2
9.53 9.91 9.16 -0.19 9.22 9.55 9.23 ~0.]3 -9.19 -.59 -0.24 9.93 -9.41 9.5B
~9.42 -9.55 09] 1 25 -9.s9 -9.59 1s 97 -9.49 -QZ] d50
1914 9.28 -9.fi1 9.33 9.14 9.4] 9.29 91fi 1] 0] -9.29 9.45 -93] 9.28 9.98 9.39
9.44 9.15 -9.9] -9.3fi 9z 049 9.9] ~0]2 -9.95 -95] 9.93 92 -9.48 9z2 ~9.48 -
9.fi1 -0.95 1.43 -9.49 69 9.29 9.23 -9.63 -0.48 d.34
1930 -9.59 Q24 -91fi Q25 -0.]1 9.23 9.99 -9.22 -9.02 9.38 9.32 9.49 9.99 -9.32
-9.9] Qzfi -9.32 -9.45 -9.14 9.39 -9.58 -9.29 9.43 9.93 9.93 9.39 9.24 9.99 -
9.54 9.15 9.44 0.94 9.41 9.16 9.44 -9.24 9.zfi 9.29 0.49 9.53
194 -.37 9.06 -9.29 -9.95 -9.36 9.28 6 9.99 -9.28 9.52 9.49 961 9.25 -041 9.96
9.19 -9.19 -9.56 -9.35 9.34 -9.39 6 9.12 -9.22 -91] 9.37 9.44 -9.1fi -9.49
9.1] 42 -0.11 926 -9.98 Q31 -9.1fi 9.41 9.93 0.36 9.58
194] 4fi -95fi 4] 9fi 9.54 91] 9.16 28 9.25 -9.31 -9.39 -9.38 92 16 9.14 9.34
9.99 9.29 -9.97 9.29 04fi 9.12 64 996 -93fi -928 9.29 -Q9 9.54 z] -9.53 0.24
1.13 -9.35 -9.53 23 z9 -9.38 -Bz9 d.41
3002 9.41 -9fi4 9.15 -9.10 5] 9.9fi -9.92 9.49 -9.11 -9.fi 9.96 -9.3fi 9.19 -
9.98 1] ~9.49 9.13 9.96 -9.14 ~9.3fi 9.35 9.35 )9 9.13 -9.69 9.29 9.1fi -9.36
9.3s 9.31 -966 0.97 d.18 -9.33 -Qfi9 1B 9.95 -048 -QZfi 0]
2010 -9.11 -9.63 fifi fifi 9.32 9.15 9.12 9.19 94] -9.12 9.11 -91] 9.11 9.12 -
9.14 ~9.31 -9.29 91] -0.22 ~9.21 91] -9.21 24 9.24 -9.33 9.18 9.1fi -Q21 9.33
9.14 -9.39 0.45 d.91 -Q24 25 -9.21 9.99 -Q28 -0.99 d.30
2011 9.33 -9.12 9.92 -9.33 9.15 9.24 -9.17 9.45 -9.04 9.99 -QZfi 9.16 Q21 -
9.98 9.33 9.19 9.5] -9.12 -0.21 9.17 9.28 -9.11 9.32 - -Q25 -9.31 --09] 9.11 -
91] 9.94 9.23 -9.93 -0.19 9.96 -9.15 -996 -9.17 9.1] -9.97 0.13 926
3018 -9.13 -9.35 922 9.53 -9.22 9.99 -9.9fi -9.12 946 -9.19 -9.19 -9.98 Q2]
99fi 9.99 9.19 -9.9] -9.93 9.91 ~9.9fi -9.22 -9.9fi 9.11 9.33 -9.19 9.15 9.13 -
99fi -9.1fi 9.23 -9.11 Bfiz 9.64 -9.93 -Q14 -9.9fi 9.9fi -9.98 0.43 d09
3U35 9.23 -9.56 936 -9.94 9.53 9.93 92 9.39 9.14 -9.zz 9.zfi -9.z3 9.15 -9.14
2s 9.45 -z4 9.15 -09] ~9.23 9.46 94B 9.45 9.11 -9.4fi 9.91 ~9.29 -Q23 fi2 9.22
042 Q23 d1fi -92] -Q50 -9.23 9.94 -9.39 -0.19 d.33
3052 37 -9.43 9.28 -9.99 9.45 9.22 -9.91 9.54 1fi -94fi -916
-.4] ~9.41 9.13 9.zfi 9.35 9.z1 9.93 -9.13 99fi fi9 9.49 ~0]1 9.31 -0.45 9.13
~9.33 -9.40 9.59 9.41 049 -0.94 d.31 -9.49 -Q58 9.14 9.24 -9.53 -0.37 d.29
2068 9.13 -9.63 5fi 9.14 9.58 9.93 9.95 9.49 9.08 -9.23 -9.99 -925 ~9.9fi -
9.11 9.13 9.49 -925 926 -9.99 9.24 947 929 ~94z -9.19 -9.48 -9.12 9.31 -9.24
1.76 9.zfi -044 0.19 1.23 -9.29 -0.42 -9.24 9.9fi -9.3z -0.25 d.34
3U]6 -9.49 56 -9.27 9.96 -0.]5 9.19 9.19 -9.44 -9.1z 9.24 9.29 9.17 9.94 -9.21
-9.91 9.38 -9.27 -9.35 -9.92 9.36 -956 -9.37 9.54 9.11 9.29 9.15 9.14 928 -967
923 9.46 -B.z9 9.14 9.27 Q46 -9.11 9.19 93z 0.15 9.34
3093 9.91 9.35 9.34 -929 -9.54 99z 9.95 9.94 -9.39 9.38 9.13 9.38 9.49 -9.45
9.99 9.55 9.94 -0.]6 -9.44 9.29 9.34 9.21 9.93 9.15 -9.91 9.24 9.26 9.18 -9.59
9.31 9.55 -0.19 9.25 -9.19 Q49 9.95 9.96 932 0.33 9.66
311] 69 922 -9.37 -9.41 9.41 9.26 9.94 -9.37 -9.36 -9.34 9.15 -9.37 9.15 9.18 -
9.9s 9.13 9.19 9.19 9.91 9.19 -9.92 -9.91 9.22 9.98 9.18 -9.35 9.11 9.94 -9.31
-0.9] -0.11 -0.30 1.39 9.96 9.28 9.47 9.32 -9.96 -0.31 d.3z
2133 9.98 -9fi9 9.1fi 9.32 9.55 9.29 9.13 9.15 9.18 9.11 9.12 99] 9.99 -9.13 -
9.29 ~9.49 -9.28 Q2 -9.95 ~9.3 92 9.5] 9.49 9.91 -9.35 9.98 9.26 -0.39 9.4
9.zfi -9.33 0.15 90] -9.35 -Q30 .21 .15 928 0.95 26
3156 -9.13 -0I5 U.]8 61 924 9.15 9.91 936 9.62 -9.14 -9.98 -9.15 9.15 9.94
9.12 ~9.42 -9.95 9.99 -0z5 ~9.1fi 9.39 -9.94 46 9.95 -94fi -0.92 9.19 -936 5B
9.26 -9.45 0.48 9.91 -9.3] -Q34 -9.13 9.ifi -9.42 -09] d2fi
315] 9.35 02 992 9.95 -9.94 9.28 -929 -9.9z 9.12 -9.1z -9.31 -9.95 9.9] -926
9.92 9.41 9.95 -9.1] -9.13 ~9.29 -9.91 9.14 9.98 9.48 9.97 -9.9] 9.28 9.fi1
9.95 -0.9] 9z] 0.18 9.35 -92z Q35 -9.29 9.29 9.59 0.25 1.16
264 -9.18 29 9.49 9.91 -9.18 9.13 91] 93fi -9.29 9.49 Q24 9.44 9.95 -9.45 4]
9] -916 -QZ] -9.18 ~9] 16 -9.1] 9.14 -924 -969 -9.24 Q25 -9.1] 1fi ~9.21 9.12 -
0.13 9.23 -9.21 Q94 -9.1] 9.94 9.91 0.32 9.1
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9.53 9.93 9.41 -9.19 9.6 9 ]9 9.22 -9.49 -9.9B 9.79 -9.25 9.39 9.17 9.29 -9.21
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9.33 9.98 9.13 0.99 9.39 -9.91 Qz1 -9.13 9.64 992 0.36 9.fi9
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9.98 -9.z9 9.98 9.57 0.12 9.66 -9.99 9.50 -9.9B 9.29 9.27 6.81 9.44
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9.52 -0.95 1 24 9fi9 -9.49 9.96 9.19 -9.38 -QZ] d.40
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9fi4 -9.9s 9.48 -9.98 0.86 1.0 -9.15 -9.91 923 963 -9.99 9.41 -09fi 9.12 -9.15
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9.16 9.42 9.36 9.17 -9.91 9.22 100 9.41 ~9.44 9.98 -9.33 -9.12 ~~9.38 -922
9.6B 9.z9 -9.49 -0.12 9.32 -9.27 9.32 -9.22 9.22 -9.29 -0.39 31
2]14 9.12 -9.08 -9.21 fi 3fi ~9.99 -99fi 9.35 9.09 -9.19 -9.19 -9.98 922 -9.19
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9.33 ~9.12 9.22 -09fi 9.31 -9.15 91] 9.47 9.58 9.97 0.41 91]
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9.19 -9.99 0]2 -9] -Q29 -0.15 9.96 -9.22 -9.96 9.31 946 956 -0.12 9.13 1.06 -
91fi ~9.98 9.52 -0.11 d99 -9.98 0]6 -9.9fi 9.96 0.93 -0.99 d.09
3886 9.92 -9.39 Sfi 9.90 3] ~9zz 916 66 9.15 -9.zfi -9z4 -9.z1 9.3z -9.94 9.23
9.37 -9.18 9.z1 9.9] 16 9.fi8 9.59 9.39 9.23 -9.23 -9.92 9.33 -91fi 1.U0 9.21 -
9z9 0.24 d.23 -9.29 -24 -9.1fi 9.1 6 9.z1 -0.22 1.22
3936 -9.54 9.38 -9.14 9.92 -9.30 ~9.94 -9.98 -9.3fi -9.01 -9.11 -9.98 -9.98 05
65 -9.9] 9.33 -9.98 0.]3 0.90 -9.98 9z9 99B 0]] -9.17 9.43 -0.95 9.12 -9.98
9.21 1.00 -916 0.97 .99 0.94 Q18 B 9.99 -9.11 0.93 d1z
2953 9.95 9.54 -9.39 -Q29 -9.40 ~9.1fi -9.11 -9.24 1 27 9.59 -9.18 9.56 9.24 -
936 -9.1fi 0.]] -9.13 -9.52 -0.27 9.46 -9.49 -9.11 9.33 9.99 9.43 -0z3 92z
9.52 -9z9 9ifi 1UU -0.91 9.41 -9.15 0.88 -9.11 9.11 073 9.52 9.24
3024 -9.99 -9.44 9.34 Q20 -9.93 ~9.15 -9.11 9.18 9.1fi 9.9] -9.16 9.11 9.9z
9.31 -9.1s 9.98 -91z -9.95 -0.99 9.11 -9.12 -9.11 9.98 9.19 -9.97 -9.92 9.96 -
Q11 9.z4 9.97 -9.91 1.UU 9.55 -9.19 -Q96 -9.11 9.11 -9.94 0.44 01
3099 9.95 9.54 -9.z] -9.11 -9.41 ~9.14 -9.14 9.15 1 06 9.43 -9.19 9.59 9.27 -
9.38 -9.16 U.79 -9.15 -9.49 -0.39 9.14 9.32 -9.14 9.34 Q22 9.4] -0.99 91] 0.7
6 -9.z4 ~9.18 U88 -0.9fi 9.33 -9.14 1.06 -9.14 9.98 U88 0.43 9.34
31]0 9.39 90] -9.21 -91fi 9.15 ~9.99 -9fi -9.31 -9.15 -9.19 -9.19 -9.98 9.24
922 -9.99 9.92 -99] 9fi 997 9.96 -9.22 -99fi 9.31 923 9.18 -012 947 -99fi -
91fi ~9.98 -9.11 -0.11 99 -9.98 -9.14 1.00 9.9fi -9.98 -0.99 d09
31]i -9.3 -9.23 9.91 9.z0 -9.22 0.96 -0.9fi -9.22 9.03 927 947 9.31 912 -9.11
9.44 9.34 9.99 -9.19 -9.98 -0.9fi -9.22 -9.9fi -0.9z -9.19 -9.16 9.34 9.s8 -
9.96 -9.1fi 9.99 -9.11 -0.11 -.99 -9.95 -98 -9.9fi 1.UU -9.98 -0.99 d.09
31]2 2 0.]] -0 7 0.93 -0 1 0.]3 -0 0 0.88 -D.DB b 1.00 0
CA 02692445 2009-12-29
WO 2008/000918 PCT/F12007/050405
204
Table 32. Discriminant Function Analysis Summary,Step 10, N of vars in model:
10; Grouping: Dfdegr (3
grps) Wilks' Lambda: .00021 approx. F(20,10)=34.077 p<.0000
WilksUpos; Partial :'=.F-remove. p-level Toler. '=. 1-Toler.
---
;2028 0.000356 0.257847;;7.1957 0 .033760 0.076143 0.923857;;
...........:.........................._:.....................................
:................ _....................................,
________ __ ........
________________________________________________________________________
1393:';0.003356 0.027331;;88.9709 0.000123 0.010301;;0.989699
_________ _ ________________________________ ______________________
_______________________________________________ _ .....................
....................... 1825 0.000415 Zz0.221021 ;8.8112 Ø022966Ø074734
0.925266
Ã1419 0.000623 0.147184:;14.4856 0.008311 0.115077;;0.884923:;
...........: ........................_:.....................................
:................ _.....................................
1688i;0.000816 0.112369;;19.7481 0.004233 0.036621;;0.963379;'
......... ................................ ...................... __
_____________________ _ _____________________ _
...................._____________________,
1540 0.004796 0.019125;128.2203 0.000051 !0.005699 0.994301
Ã1905 0.000941 0.097453:;23.1533 0 .002965 0.015289 0.984711:;
892 ;;0.001987 0.046168;;51.6506 0.000458 0.006722;;0.993278;;
............. ________________________ _______ _______ ......................
________ _________ _: 1095 0.001122 0.081712 ;28.0953 0.001909 0.023183
0.976816
1054 0.000443 Z0.206883:;9.5841 0.019467 0.033402 0.966598;;
Table 33: p-Levels for Pairwise Comparison of Dependent Variable
hESC EB St3
-------------------------------- , --------------------------------------------
- ,--------------------------------------------- _-----------------------------
------------ -
hESC 0.000030 ?0.001469
:,__,_,__,_, EB _,__,_,__,_,;______________________________________________
________ ...................................... 0.000030 ?0.000004
_____________St3 ;,_,_
0
;0.001469 .000004
Table 34. Chi-Square Tests with Successive Roots Removed
---- ---- ---- ----: -- ---- ---- ---- ---. ---- ---- ------------------- -----
--------------- ------ .-----------------
Eigen- Canonicl;:Wilks' Chi-Sqr.:';df;: p-level
=,_,__,_,__,_,__,_,__,::__,_,__,_,__,_,__,_,__,_,__,_,,_,_,__,_,__,_,__,_,___,_
_,_::_,_,__,_,__,_,__,_,_;
_....................
0 ;543.6531=;0.999082;;0.000092 ?88.31998=;20;0.000000
................................,:..............................;..............
....... .....,; ...................,i
..... ...........
1,;19.0192 ;0.974704;0.049952 0.46856;9 ;0.000796:;
-~ ,
Table 35. Raw Coefficients for Canonical Variables
..................... =
:.............................................
Root 1 Root 2
................................................:....................:
2028 7.5848 31.1129
---- _--
1393 -87.7220 i;-17.5404
.................... 1825 '=.-20.3737 <i1.2276
.....................................................................:
1419 -1.6112 ;0.6578
- ---- _-- ------- ------ ----------- =
1688 26.9100 i;-22 ,__.0977:;
,__õ
1540 ~'=.-23.8102 <`=.2.0084
.......................... ...................... . ....................
1905 2.4675 -1.3916
892 ~:z22.1050 '!;6.1419
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1095 19.1659'!;-10.0060
__________________________________________________
1054 -3.6582 -3.6138
...................... ____ .......... _ _________________
Constant 35.8460 ;32.4125
:........................:.......................................
Eigenval 543.6531:,19.0192 ______________________________
Cum.Prop 0.9662 1.0000
Table 36. Means of Canonical Variables
:.................................. ....................
Root 1 Root 2
.. ............. ................... . . . . . . . . ..
hESC9.6048 6.90485 ;.;
.. . . . .............. .. ...................:
EB -24.6352 -1.0695513
............... St3 22.3379 -3.35542Ã
Table 37. Five discriminative masses for embryonic stem cells, Eigenvalues,
canonical
means and raw coefficients.
............. ............................................ .... .... .... ....
.... .... .... .... .... .... .... .... .... .
............ .................... _____________________
Wilks';; Partial F-remove p-level Toler. 1-Toler. , -------------------- -
-----------------
; 892 0.037703 0.371871?8.44552 0.007112 ;0.252464:;0.747536~~
________________________________,____,____,____,____.,___________________
1540 0.076441 0.183420'22.25982 0.00020810.112403;0.887597
:.... ........ ......... ......... ,...... ......... ......... ........ ......
......,
4905 0.116818 0.120023 36.65870 M.000025 0.114965 0.885035
__________________õ________________________ _ ____________ _____________
1393;0.052729 0.26590143.80400 0.001329 ;0.236781;0.763217~~
.......... _____________________ ..................... .....................
1688 0.037126 0.377655;8.23959 0.007682;0.20256410.797436
................ .... .... .... .... .... .... .... .... .... .... .... ....
.... .... .... .... .... .... .... .... .... .....
___________________________.;_____________________________
...............
Eigen- Canonicl~Wilks'i;Chi-Sqr.::df:~ p-level
.............. ___ .........................
............... ....... _......................
;24.17569'=,0.979938 ;0.014021 ;51.20657;10:;0.000000j;
------ --------------------- ,------ __,_____________________., .; _____õ_
1 ;1.83300 ?0.804374:;0.352983 ?12.49602;4 ;0.014020';;
... .... .... .... .... .... .... .... ..
___________________ _____________________
Root 1 Root 2
hESC -1.52086;-2.17499
EB 5.10674 ;0.42329
:........................................................:
St3 E-4.94395i;0.95615
____________ ____________
Root 1 Root 2
______________ ___
892 z-2.9664 :z1.06236:;
1540 4.9385 :':0.98374
1905 -1.0331 :;-0.05027;'=,
1393 Iz16.5002z-0.73876
.. .. . ..................................................:
1688 !:-11.7267;':5.32870
Constant 15.5575 :;-2.30082:'
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:............
Eigenval 24.1757 1.83300
__________________________________________________
Cum.Prop 0.9295 1.00000
Table 38. Four discriminative masses for embryonic stem cells, Eigenvalues,
canonical
means, their p values and raw coefficients.
................ .... .... .................. ...
.......... .............................. _
___________________________________________ _____________
_.__
?Wilks'Partial ;F-remove ' p-level Toler. ? 1-Toler.
__________õ______________________________ ..................... ,
892 i0.154395 0.341522;10.60439 j0.002715~'0.330624i0.669376~;
;- -- --- ---- -- ---- - ; ---- --- ---- ---- --- ---- --- -- ---- --- -- ----
- =
4540 ;0.158162 =0.333388 10 99728 0 0023780.220196 ;0.779804
.... .....................
1905:'0.186838 0.282219:;13.98840 ;0.000951 ?0.280169 ?0.719831~;
_________________________________________ ....................
__,____,____,____, _,____, ..................... .....................
4688=i0.070024 0.753016:?1.80397 "=0.210098:i0.445690:i0.554310;
.................. .... .... ................. .... .... .... .... .... ....
.... .... .... .... ....
;-_ =-------------------- --------------------- :-----------------------------
------------------- . ---------------
Eigen- ;Canonicl:;Wilks' ;Chi-Sqr.s;dfs; p-level
0 ;5.732435:;0.922749,:0.052729 ?36.78228:;8 0.000013
_,;________________,_.,,____,____,____,___,;
;1.816924 ?0.803121;0.354997 ;12.94557:;3 ;0.004756~;
,Means zz Root 1 ; Root 2
.. .. .. ....: ................... :...................::
hESC !;0.96322 '-2.13740
................... ................... _ _________________õ
EB ;-2.52606:;0.33923
- - ---- - =- ---- ---- ---- -- = ---- ---- ---- ----=
St3 ;2.30492;1.02923
................... ....
.................................................................
______________________ .................... .....................
....................
P values ; hESC EB :zz St3
---------------------------------------------
__________________________________________
hESC 0.0016530.010579``
E
________B M.001653 ____________________
=_____________________.____________________
-
'0.000192
....................... õ___________________,,____________________ _________
St3 0.010579 ?0.000192 zz
................ .... .... .... .... .....
_______ ___________________=
...............
Root 1 Root 2
892 2.7193 1.27734
.. .......:....................:....................:
:......... . . .
1540 -3.3607 0.79112
__ _ ____________________________________
1905 0.6350 -0.01744:;
__ _______ -------------------- õ___________________
1688 3.3118 5.25235:'
:........................:....................:....................:
Constant -10.6166;;-2.94827
- - - ------=--------------------=
Eigenval 5.7324 1.81692
........................ . ................... . ................... Cum.P rop
0. 7593 1.00000
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Table 39. Factors identified for combined neutral and acidic glycans.
24.40 11.75 10.76 8.22 7.00 6.06 5.41 5.27
Fal Fa2 Fa3 Fa4 Fa5 Fa6 Fa7 Fa8
609 -0.57 0.11 0.07 0.32 0.12 -0.02 0.07 0.13
730 0.12 -0.15 -0.30 -0.69 0.00 -0.20 0.17 0.01
771 0.56 -0.01 -0.08 -0.52 -0.31 0.03 0.19 0.03
892 0.68 0.01 -0.23 -0.55 -0.10 -0.10 -0.10 -0.13
917 0.30 0.07 0.18 -0.53 -0.57 0.21 0.28 -0.03
933 0.68 0.17 0.14 -0.05 -0.47 0.10 0.06 -0.04
1031 -0.08 0.02 -0.08 -0.55 0.05 0.04 0.08 -0.04
1054 0.64 0.02 -0.19 -0.36 -0.22 0.03 -0.05 -0.11
1079 0.35 0.32 0.20 -0.47 -0.56 0.18 0.16 -0.07
1095 0.72 0.15 0.24 0.02 -0.31 -0.25 0.14 -0.07
1120 0.23 -0.16 -0.20 -0.30 -0.85 -0.06 0.14 0.05
1136 0.12 0.02 -0.50 -0.10 0.14 -0.77 -0.19 -0.03
1209 0.09 -0.08 -0.24 -0.20 0.00 -0.88 -0.07 0.02
1216 0.91 -0.14 -0.03 -0.10 -0.01 -0.15 0.02 -0.11
1241 0.21 0.12 0.38 -0.13 -0.80 -0.21 0.19 0.12
1257 0.08 0.55 0.27 -0.10 0.07 0.24 0.58 -0.01
1282 -0.01 0.08 -0.17 -0.37 -0.78 -0.12 -0.07 -0.04
1298 0.15 0.46 -0.01 0.33 0.61 -0.21 0.02 -0.11
1323 0.04 -0.24 -0.18 -0.25 -0.76 0.24 0.00 0.07
1339 0.03 -0.17 -0.22 -0.33 -0.74 0.36 0.07 0.02
1378 0.91 -0.11 -0.08 0.17 -0.30 -0.02 0.06 -0.09
1393 -0.25 0.10 0.20 0.23 0.14 -0.17 0.47 0.06
1403 0.14 0.15 0.18 -0.28 -0.79 0.12 0.25 -0.02
1419 -0.17 -0.22 0.87 0.27 -0.05 0.19 0.09 0.08
1444 -0.01 0.17 0.02 0.17 -0.71 -0.03 -0.03 0.43
1460 0.11 0.67 -0.35 0.20 0.49 0.18 -0.01 -0.18
1485 -0.17 0.69 0.00 -0.41 0.44 -0.06 -0.17 -0.18
1501 0.11 -0.14 -0.24 -0.39 -0.70 0.35 -0.03 0.12
1540 0.91 -0.17 -0.29 0.12 0.11 -0.06 0.07 0.04
1555 0.06 0.04 0.44 -0.13 0.26 -0.05 -0.16 0.35
1565 0.11 0.12 0.26 -0.77 0.01 0.02 0.43 0.01
1581 -0.54 -0.47 0.59 0.07 0.02 0.00 0.03 0.24
1590 0.15 -0.30 0.00 0.03 0.14 -0.56 0.28 0.09
1606 -0.19 0.82 0.21 -0.03 0.15 -0.23 -0.11 -0.22
1622 0.11 0.67 -0.40 0.22 0.41 0.22 0.13 -0.19
1647 -0.41 0.73 0.22 0.03 0.22 -0.34 0.13 -0.16
1663 -0.50 0.24 -0.29 0.49 0.21 -0.21 -0.06 -0.20
1688 -0.22 0.26 0.17 -0.74 0.00 -0.21 0.28 -0.31
1702 0.93 -0.07 -0.24 0.00 -0.06 -0.21 0.09 -0.07
1704 -0.09 0.90 -0.06 0.14 0.00 0.11 -0.22 -0.24
1717 0.06 -0.22 0.28 -0.15 0.21 -0.52 0.07 -0.32
1743 -0.67 -0.49 -0.02 0.09 0.40 0.06 -0.15 0.16
1768 -0.22 0.31 -0.29 -0.24 0.10 -0.02 0.03 0.39
1784 0.08 0.11 0.06 0.10 0.07 -0.01 -0.28 -0.80
1793 0.18 -0.36 -0.27 0.04 -0.73 0.10 0.08 0.04
1809 -0.59 0.20 0.05 0.50 0.06 0.03 0.06 0.02
1825 -0.16 -0.03 -0.31 -0.73 -0.10 0.44 -0.05 0.00
1850 -0.10 0.74 -0.25 -0.31 0.02 -0.03 0.22 -0.24
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1866 -0.01 0.74 -0.27 0.02 0.13 0.38 -0.22 -0.29
1905 -0.26 -0.42 -0.41 0.33 0.34 0.03 -0.55 0.00
1955 -0.66 -0.32 -0.02 0.24 0.21 -0.14 0.17 0.18
1971 -0.12 0.55 -0.26 0.01 0.01 -0.40 0.45 -0.03
1987 0.10 -0.29 -0.57 0.12 -0.63 0.16 -0.08 0.02
1996 0.05 -0.14 -0.14 -0.42 -0.75 0.22 0.21 0.04
2012 0.11 0.79 -0.03 0.25 0.13 -0.37 -0.09 -0.19
2028 -0.57 0.02 0.26 0.44 0.25 -0.25 -0.22 0.14
2041 -0.24 0.22 0.18 0.31 0.29 -0.65 -0.41 0.06
2067 0.01 -0.32 0.06 0.45 0.45 0.05 -0.64 -0.02
2101 -0.26 -0.24 0.24 0.34 -0.58 -0.22 0.22 0.17
2117 -0.01 -0.03 0.20 -0.54 0.10 0.07 0.12 -0.04
2142 0.40 -0.21 -0.06 0.24 0.48 0.14 0.17 0.12
2158 0.18 0.03 -0.17 -0.08 0.06 -0.73 0.35 0.03
2174 -0.61 -0.05 0.26 0.45 0.23 -0.19 -0.10 0.04
2229 -0.02 0.46 0.12 0.21 0.18 -0.42 -0.52 -0.27
2304 0.05 -0.05 0.17 -0.89 0.00 -0.16 0.26 0.00
2320 -0.28 -0.31 0.09 0.10 0.10 -0.42 -0.08 0.12
2391 0.07 0.09 -0.22 -0.10 0.07 -0.76 0.25 0.04
2393 0.10 -0.02 -0.21 -0.14 -0.01 -0.06 0.08 0.02
1354 -0.11 0.10 -0.83 -0.12 -0.07 -0.42 0.05 0.00
1362 0.40 -0.12 -0.05 0.10 0.25 0.09 0.14 0.01
1475 0.02 -0.07 0.00 -0.87 -0.35 -0.04 0.10 -0.01
1500 0.17 -0.28 -0.46 -0.46 -0.35 -0.37 0.11 0.08
1516 0.14 0.07 -0.45 -0.12 0.07 -0.67 0.18 0.03
1541 0.02 -0.18 -0.89 -0.16 0.00 -0.21 -0.11 0.00
1549 -0.19 -0.29 0.15 0.28 0.27 0.07 0.02 0.20
1557 0.02 0.23 -0.74 0.21 0.08 0.40 0.23 -0.02
1565 -0.06 -0.18 -0.24 0.15 0.40 -0.15 0.54 0.21
1637 0.10 -0.09 -0.01 -0.90 -0.31 -0.09 0.12 0.01
1678 -0.02 0.05 -0.11 -0.61 -0.25 -0.08 0.70 0.12
1703 0.43 -0.09 -0.53 -0.12 -0.41 0.03 0.23 0.03
1711 0.35 0.08 0.65 -0.23 -0.14 0.06 -0.03 -0.20
1719 0.41 0.06 -0.66 0.21 -0.44 -0.16 0.30 0.06
1727 -0.18 0.00 -0.01 -0.34 0.38 0.02 0.72 0.15
1744 0.07 -0.02 -0.22 -0.53 -0.02 -0.28 0.14 0.03
1768 0.17 0.28 -0.24 0.13 0.17 0.04 0.52 0.01
1791 0.00 -0.12 -0.78 -0.21 -0.03 -0.51 -0.14 0.01
1799 -0.13 -0.01 -0.17 -0.85 -0.04 0.42 0.07 -0.03
1840 -0.12 0.14 0.18 -0.57 0.08 0.24 0.49 0.04
1865 0.36 -0.11 -0.86 -0.12 0.03 0.03 0.21 0.05
1873 0.02 -0.01 -0.13 -0.34 -0.17 0.12 0.80 0.11
1889 -0.08 0.01 -0.27 -0.04 -0.11 0.08 0.89 0.19
1906 0.38 -0.17 -0.31 -0.59 -0.14 -0.13 0.45 0.09
1914 0.42 -0.39 -0.05 -0.26 -0.06 -0.12 0.66 0.19
1930 -0.35 0.01 0.04 0.63 0.52 0.19 0.01 0.08
1946 -0.22 -0.43 0.18 0.37 0.37 0.11 0.08 0.19
1947 0.17 0.09 -0.26 -0.55 -0.38 -0.25 0.35 0.02
2002 0.20 0.03 0.13 -0.51 -0.19 -0.18 0.64 0.06
2010 0.00 0.10 -0.83 -0.25 -0.01 -0.13 0.19 0.01
2011 0.06 -0.20 0.15 -0.01 -0.64 0.07 0.03 0.11
2035 0.22 0.08 0.00 -0.63 0.08 -0.23 0.41 0.04
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2052 0.12 -0.26 0.04 -0.32 -0.28 -0.10 0.54 0.15
2068 0.10 0.02 -0.46 -0.73 -0.01 -0.16 0.20 0.02
2076 -0.19 0.00 0.30 0.67 0.45 0.22 -0.15 0.06
2092 -0.24 -0.27 0.48 0.56 0.12 0.01 -0.02 0.45
2117 0.15 -0.02 0.71 -0.21 -0.29 -0.04 0.18 0.03
2133 0.03 0.01 -0.31 -0.69 0.17 0.06 0.34 -0.01
2156 0.11 -0.02 -0.88 -0.19 -0.06 -0.16 0.33 0.08
2157 0.01 0.80 0.06 -0.08 -0.17 0.15 -0.12 0.37
2164 0.16 -0.20 -0.01 0.05 0.11 -0.01 0.14 0.09
2221 0.03 0.14 0.12 0.20 0.29 0.05 -0.13 -0.84
2222 -0.31 0.00 0.39 0.37 0.05 0.02 -0.47 0.60
2230 0.37 -0.09 -0.70 -0.15 -0.42 0.02 0.19 0.06
2237 -0.07 -0.25 0.14 0.32 0.45 0.03 0.22 0.13
2238 0.24 0.05 0.12 0.01 -0.42 0.08 -0.28 0.60
2239 0.42 -0.27 0.09 0.06 -0.41 0.09 0.19 0.04
2253 0.00 -0.21 -0.04 0.26 0.37 0.13 0.05 0.08
2254 -0.13 -0.15 0.02 0.09 0.12 0.06 -0.18 0.07
2263 -0.16 -0.25 0.60 -0.05 -0.20 -0.09 -0.16 0.50
2279 0.25 0.12 0.04 -0.43 -0.02 0.05 0.08 -0.76
2280 0.05 0.20 0.16 -0.50 0.24 -0.14 0.16 -0.60
2295 -0.06 -0.52 0.00 0.34 0.42 0.08 0.35 0.20
2321 0.02 -0.18 0.09 0.15 -0.75 -0.01 -0.02 0.06
2367 -0.25 0.28 0.39 0.48 0.26 0.09 -0.55 -0.22
2368 -0.27 0.35 0.24 0.19 0.20 -0.11 -0.23 0.57
2383 -0.33 -0.32 0.24 0.28 0.17 0.04 0.01 0.18
2384 -0.29 -0.35 0.16 0.33 -0.32 0.00 0.06 0.11
2390 -0.49 -0.16 0.16 0.33 0.27 0.03 -0.25 0.39
2400 0.21 -0.17 0.28 0.04 -0.44 -0.14 0.10 0.00
2408 -0.04 0.09 0.35 0.38 -0.18 0.10 -0.48 0.53
2425 0.07 0.09 0.34 -0.45 -0.53 -0.06 -0.02 0.51
2441 -0.15 0.00 0.49 0.29 0.09 0.17 -0.71 -0.11
2447 0.25 0.03 -0.72 0.07 0.06 -0.16 0.31 0.05
2448 0.01 0.24 -0.71 0.23 0.08 0.36 0.40 -0.01
2482 0.00 -0.13 -0.05 -0.76 -0.44 -0.07 0.15 0.01
2512 0.58 -0.14 -0.13 0.11 0.38 0.17 0.12 0.02
2521 -0.08 -0.31 -0.29 -0.09 -0.27 -0.21 0.20 0.11
2522 0.03 0.17 -0.66 0.21 -0.26 0.43 0.24 0.00
2528 -0.08 -0.17 0.03 0.11 0.14 0.06 -0.13 0.07
2529 0.39 -0.18 0.08 0.16 0.25 0.12 0.32 0.04
2544 -0.12 -0.15 0.01 0.10 0.12 0.07 -0.18 0.07
2570 -0.14 0.34 0.04 -0.07 0.06 0.10 0.04 -0.77
2571 0.15 0.13 -0.04 0.06 -0.37 -0.39 -0.16 -0.69
2586 0.63 -0.14 0.02 0.09 0.23 0.10 0.16 0.07
2587 -0.32 0.16 0.27 0.18 -0.06 0.01 -0.52 0.65
2603 0.32 -0.13 0.10 0.23 -0.82 0.09 0.00 0.04
2644 0.12 0.13 -0.11 -0.33 -0.14 0.08 -0.10 -0.86
2645 0.08 0.17 0.14 0.01 0.07 0.02 -0.20 -0.90
2683 0.25 -0.28 -0.28 -0.21 -0.46 0.08 0.16 0.04
2732 -0.16 0.17 0.11 0.38 0.28 0.00 -0.62 -0.52
2733 0.02 0.38 0.13 0.04 0.28 0.04 0.07 0.39
2807 -0.07 0.20 0.26 0.10 -0.01 -0.04 -0.75 -0.17
2878 -0.13 -0.06 0.04 0.05 0.34 0.15 0.26 0.04
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2879 0.26 -0.24 0.08 0.05 0.31 0.02 0.01 0.04
2886 0.11 -0.13 -0.45 -0.46 0.02 -0.30 0.01 0.01
2936 -0.01 0.34 0.09 0.24 0.10 0.03 -0.19 -0.87
2953 -0.35 0.06 0.25 0.24 0.14 0.05 -0.53 0.44
3024 -0.17 0.45 -0.35 0.06 -0.03 -0.65 0.22 0.02
3025 -0.40 0.43 -0.05 0.22 -0.02 -0.01 0.20 0.04
3098 -0.07 0.12 0.16 0.22 0.12 0.01 -0.32 -0.87
3099 -0.28 0.13 0.03 0.24 0.14 0.18 -0.68 0.48
3172 0.00 0.41 0.18 0.11 0.09 0.07 -0.70 0.47
3390 -0.41 0.26 -0.01 0.18 -0.02 -0.01 0.06 0.05
3463 -0.53 -0.27 0.13 0.22 0.12 -0.03 -0.07 0.09
Expl.Var 15.087 13.057 17.101 19.989 17.715 9.359 14.429 12.390
Prp.Totl 0.093 0.080 0.105 0.123 0.109 0.057 0.089 0.076
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Table 40. Raw Canonical Discriminant Function Coefficients, Eigenvalues,
Means, Tests of
Significance of Squared Mahalanobis Distances and Classification Matrix for
acidic glycans from
embryonic stem cells.
..................................
...............................................................................
..................................................
.............................
____________________ ___________________ _____________________________
_____________________________ __________________ _____________________
___ ___
Wiiks&apos, Partial F-remove p-level Toler. 1-Toler.
............ ........
2092 0.000224 0.012148 203.3029 =Ø000016 0.014747 0.985253
...................
............. . . . ... .
2222 10.000179 0.015219 1161.7677 0.000029 0.006831 10.993169
, ---
--- _- -- ---:
3463 0.000076 0.035969 67.0037 0.000245 0.011522 0.988478
2383 _______________________;
0.000102 0.026735 191.0094 0.000117 0.014976 0.985024
...................
........................................................... ...............
2482 0.000099 0.027361 88.8701 0.000124 0.013292 0.986708
2237 10.000080 0.034031 70.9618 0.000214 0.019852 ,0.980148
_____ ___...................................................
................................. ................................
...........................
2408 0.000052
0.052279 45.3200 0.000625 0.006492 0.993508
...... ....._ .................... ;...................
;.................................,.................................,..........
........
1678 0.000040 ................
0.068113 134.2039 0.001211 0.021660 0.978340
, _. .____.___ ........................... 2368 0.000011 0.248367 7.5658
0.030742 0.147828 0.852172
__ ...................................... õ................................
............................
1703 0.000010 `;0.273081 6.6548 0.038970 0.142395 Ã0.857605
...........
p-levels (Stem cell ACIDIC ES BM CB CD133 CD34 v03)
................................... ; ___.____.____.____.____.____.____.,
hESC EB st3
...____._
hESC '0.000001 0.000000
EB 10.000001 0.000005
st3 '0.000000 0.000005
................................... ..................
.............
Classification Matrix
Percent hESCõEBi;st3
------ ------------------ ________
hESC 100.0000i4 0 ,0
_______________________
EB 1100.0000;0 i7 '=,0
.............................................................. ........ .
st3 100.0000;;0 ;:6
------------- ----------------------- -------------- ______
Total;100.OOOO4 7 6
Chi-Square Tests with Successive Roots Removed (Stem cell ACIDIC ES BM CB
CD133 CD34 v03);
Eigen- Canonicl Wilks' Chi-Sqr. df p-level
0 11926.162 ;0.999741 ;0.000003 121.7442 20 0.000000
............. ............ ............ ................................
1 189.829 ;0.997376 ;0.005240 49.8881 9 0.000000
Raw Coefficients (Stem cell ACIDIC ES BM CB CD133 CD34 v03) for Canonical
Variables;
---- = ------------------------------------------------------------------ -----
----------------------------------------------------------
Root 1 Root 2
... ......... ......... ......... ......... . .. . ......... .........
......... .. . . ......... ......... ........... ;
2092 zz3.556 ? 14.963
_________________________________________________________________________
2222 '3.870 '-5.504
...........
...... .........._ ........... ..........._>;
_______________________________________________________________________________
__________________________________________
3463 ;124.635 ;-123.334
- - ---- -- ---- ---- --- ---- - ---- -- - -- - -- ---- - ---- --- --- ---- ---
---- --- --- ---- - ---- ---- ;
2383 :z3.237 ?-19.567
_________________________________________________________________________
2482 :-17.091 ;-3.553
...........
_____________________________________________________________
___...................................................... 2237 ;9.232 ;2.593
- - ---- -- ---- ---- --- ---- - ---- -- - - - -- ---- - ---- --- --- ---- - -
---- --- --- ---- - ---- -
2408 A7.326 15.436
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1678 8.774 3.763
.......... ........... ........... ........... . ........... .........._
........... ..........._>;
__________________________________________________________________
.............................................................. 2368 1.257
2.746
.......... ........... ........... ............:;
..............................................................
.
1703 -3.675 0.679
...
Constant -41386 -8.778
.......................
..............................................................
..........................................
.......... .... ...._.. ........... . ........... .......... _
_________________________________. _________________________
....................... Eigenval 1926.162 189.829
.......... ........... ........... ............:;
p _ _ _ : _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _.
Cum.Pro 0.910 11000
Means of Canonical Variables (Stem cell ACIDIC ES BM CB CD133 CD34 v03);
........................................................... ________
Rootl Root2
....................................... _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ __ _ _
õ_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _. ;
hESC ~66.2050 :;-8.7238
. ....................................... .... .... .... .... .... ..>....
.... .... .... .... .... .... .... .... .... .... .... ...._::
...........
.................................................................... EB ;-
4.0512 14.8899
- - ---- -- ---- ---- ---- - ---- -- ---- ---- -- ---- - - --- --- ---- -- ----
- ---- --- -
st3 ,39.4102 11.5557
........................................ .... .... .... .... .... : >.. ....
.... .... .... .... .... .... .... .... .... ..............
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Table 41. Raw Canonical Discriminant Function Coefficients, Eigenvalues,
Means, Tests of
Significance of Squared Mahalanobis Distances and Classification Matrix for
combined neutral and
acidic glycans from embryonic stem cells.
Raw Canonical Discriminant Function Coefficients (NEUTRAL and ACIDIC) Sigma-
restricted parameterizatior
Function Function
.. ......... ......... ........ ......... ......... ,....... .........
......... ......... ......... ........ ...... ......... ......... .........
Intercept ;-171.07 109.808
....... ........... ........... ........... ............ . ..........
........... ............
"730" -20.64 :-3.629
....... ........... ........... .......... ... ....
_______________________________________________________________________________
________________
_______________________________________________________________________________
_________________________________________________________________________
"1095" -41.12 z32.862
... ......... ......... .. .. . ......... ......... . .. . ......... .........
......... ......... . .. . ......... ......... ......... .........
"1540" 19.51 ':= 1.698
:... ... .......... ...........
"1850" -2.59 52.608
_______________________________________________________________________________
___________________________
"2174" 312.41 12.862
... ......... ......... .. .. . ......... ......... . .. . ......... .........
......... ......... . .. . ......... ......... ......... .........
"1799" 43.37 ':14.909
:... ... .......... ...........
"2092" -7.76 :8.021
:......
-- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---------------------
--------------- ---------------------------------------------------------------
-------------::----------------------------------------------------------------
------------
"2222" 40.25 z2.177
.. ......... ......... .. .. . ......... ......... . .. . ......... .........
......... ......... . .. . ......... ......... ......... .........
"2230" 27.50 =8.883
....... ........... ........... ........... ............ . ..........
........... ............
"2237" 37.11 ;7.990
= .... ....
"2280" ;11.20 z-3.048
... ......... ......... .. . .. ......... ......... . .. ......... .........
......... ......... . .. . ......... ......... ......... .........
"2441 " -1.64 s':= 1. 517
:... ... ..........
_
"2587" ; 19.79 -12.728
..> ....... ........... ........... ..........
Eigenvalue 33714.84 177.818
... ......... ......... .................. ......... ,....... .........
......... ......... ......... ........ ......... ......... ......... .........
Cum. Prop. 0.99 1.000
:... ... .......... . ...... ...
Chi-Square Tests with Successive Roots Removed Sigma-restricted
parameterization;
Eigen- Canonicl Wilk's Chi-Sqr. df p-level
__ ---------------------------- ----------------------------- õ----------------
-------------------------- ____________________
0 33714.84 0.999985 0.000000 124.8967 ?26.00000 0.000000
.................. ............. ............................. ....... 1
177.82 '=,0.997200 0.005592 41.4909 112.00000 0.000041
Class Means for Canonical Variables Sigma-restricted parameterization
________________________________________
________________________________________________
hESC EB st3
_1 ________________________________________
_________________________________________ ...........................
______________________
,296.9877 <,-64.8879 =-122.289
__________________________________________
_________________________________________ .....................
__________
2 3.2762 13.6745 ;-13.769
Tests of Significance of Squared Mahalanobis Distances
F tests with 13 and 2. degrees of freedom Sigma-restricted parameterization
________
hESC hESC EB EB st3 st3
_____________________ _________
hESC ? ?3671.084 0.000272 4639.207 0.000216
............... _____ .......... EB ;3671.084 0.000272 143.719 z0.006930
..._. ____________________ _.__._._ ___ .........................
......................... __.____.____.____.___
st3 ;4639.207 '0.000216 !Ã143.719 0.006930
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Classification Matrix Rows: Observed classifications Columns: Predicted
classifications
__________________
Percent hESC EB st3
: _________________________________________,_
hESC 100.0000 4.000000 0.000000 ?0.000000
....... ........ .........
EB 100.0000 :0.000000 7.000000 0.000000
........._ :.. .... .... ..........................._
:................................. .... _;,.... .... .... .... .... .... ....
...._:,. .... .... .... .... .... .... .... .._;;
............ ___......................... ...................................
__________ _
st3 100.0000 0.000000 0.000000 6.000000
.......................................... :;
Total 100.0000 Z4.000000 7.000000 6.000000
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Table 42. m/z.= neutral=[M+Na]+, sialylated=[M-H] ; Composition: S=NeuAc,
G=NeuGc,
H=Hex, N=HexNAc, F=dHex; ST (structure class): M=mannose-type, H=hybrid-type,
C=complex-type, O=other.
Neutral N-glycan fraction (Fig. 2012 2012,72 H5N5F1 C
1.A 2028 2028,71 H6N5 C
Fig. m/z Composition ST 2067 2067,69 H10N2 M
609 609,21 H1N2 M 2101 2101,76 H5N4F3 C
771 771,26 H2N2 M 2142 2142,78 H4N5F3 C
917 917,32 H2N2F1 M 2174 2174,77 H6N5F1 C
933 933,31 H3N2 M 2229 2229,74 H11N2 M
1079 1079,38 H3N2F1 M 2304 2304,84 H5N5F3 C
1095 1095,37 H4N2 M 2361 2361,87 H5N6F2 C
1120 1120,40 H2N3F1 H Sialylated N-glycan fraction (Fig.
1136 1136,40 H3N3 H 1=B
1241 1241,43 H4N2F1 M Fig. m/z Composition ST
1257 1257,42 H5N2 M 1565 1565,55 S1H4N3 0
1282 1282,45 H3N3F1 H 1678 1678,60 S2H2N3F1 0
1298 1298,45 H4N3 H 1711 1711,61 S1H4N3F1 H
1323 1323,48 H2N4F1 C 1727 1727,60 S1H5N3 H
1339 1339,48 H3N4 C 1768 1768,57 S1H4N4 C
1403 1403,48 H5N2F1 M 1799 1799,62 S2H4N2F1 0
1419 1419,48 H6N2 M 1840 1840,65 S2H3N3F1 H
1444 1444,51 H4N3F1 H 1873 1873,66 S1H5N3F1 H
1460 1460,50 H5N3 H 1889 1889,65 S1H6N3 H
1485 1485,53 H3N4F1 C 1914 1914,68 S1H4N4F1 C
1501 1501,53 H4N4 C 1930 1930,68 S1H5N4 C
1542 1542,56 H3N5 C 1946 1946,67 G1H5N4 C
1565 1565,53 H6N2F1 M 1971 1971,71 S1H4N5 C
1581 1581,53 H7N2 M 2002 2002,70 S2H4N3F1 H
1590 1590,57 H4N3F2 H 2035 2035,71 S1H6N3F1 H
1606 1606,56 H5N3F 1 H 2076 2076,74 S 1 H5N4F 1 C
1622 1622,56 H6N3 H 2092 2092,73 G1H5N4F1 C
1647 1647,59 H4N4F1 C 2117 2117,76 S1H4N5F1 C
1663 1663,58 H5N4 C 2133 2133,76 S1H5N5 C
1688 1688,61 H3N5F1 C 2164 2164,75 S2H5N3F1 H
1704 1704,61 H4N5 C 2221 2221,78 S2H5N4 C
1743 1743,58 H8N2 M 2222 2222,80 S1H5N4F2 C
1768 1768,61 H6N3F1 H 2237 2237,77 G1S1H5N4 C
1793 1793,64 H4N4F2 C 2238 2238,79 S1H6N4F1 C
1809 1809,64 H5N4F1 C 2253 2253,76 G2H5N4 C
1825 1825,63 H6N4 C 2263 2263,82 S1H4N5F2 C
1850 1850,67 H4N5F3 C 2279 2279,82 S1H5N5F1 C
1866 1866,66 H5N5 C 2295 2295,81 S1H6N5 C
1905 1905,63 H9N2 M 2367 2367,83 S2H5N4F1 C
1955 1955,70 H5N4F2 C 2368 2368,85 S1H5N4F3 C
1987 1987,69 H7N4 C 2383 2383,83 S2H6N4 C
1996 1996,72 H4N5F2 C 2384 2384,85 S1H6N4F2 C
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2408 2408,86 S2H4N5F1 C
2425 2425,87 S1H5N5F2 C
2441 2441,87 S1H6N5F1 C
2482 2482,90 S1H5N6F2 C
2570 2570,91 S2H5N5F1 C
2571 2571,93 S1H5N5F3 C
2587 2587,93 S1H6N5F2 C
2603 2603,92 S1H7N5F1 C
2644 2644,95 S1H6N6F1 C
2732 2732,97 S2H6N5F1 C
2733 2733,99 S1H6N5F3 C
2807 2807,00 S1H7N6F1 C
2878 2878,00 S3H6N5 C
2879 2879,02 S2H6N5F2 C
2953 2953,06 S1H7N6F2 C
3098 3098,10 S2H7N6F1 C
3099 3099,12 S1H7N6F3 C
3172 3172,13 S1H8N7F1 C
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Table 43. Comparison of lectin ligand profile in hESCs and MEFs
a a
Lectin hESC MEF a a
...................................................,...........................
..................... ,.......................................;
a a
PSA - + :..
..................................................~............................
......................; .. .....................................~
-
MAA +
;...................................................;,.........................
........................;......................................~
-
PNA +
a a
RCA + + present in cell surface
- not present in cell surface
Table 44. Lectins
ES29 FES30
PSA - -
LTA +/- -
EA -
MAA +
SNA (+/-) (+/-)
RCA + +
PNA +
PWA +
STA
WFA +
PHA-L (+/-) (+/-)
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Table 45. FACS
ES30 FES61
PSA +
LTA +/-
EA +
MAA +
SNA
RCA
PNA +
PWA /- -
STA +/- +/-
WFA
PHA-L
1PA + +/-
MBL - -
Table 46. Antibodies
Immuno FAC S
GF281 -
GF285 - -
GF286 +/- +
GF287 + +
GF372 -
GF373 -
anti-Le -
GF368 +/- -
GF279 + +
GF280 -
GF284 +/- -
GF288 +/- -
GF289 (+/-) -
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Table 47. Antibodies
mmuno ACS
GF403 -
GF418 -
anti-Le x -
anti-sialyl
Le x
GF369 /- -
GF370 /- -
GF371 -
GF367 1/
GF401 - -
GF283 /-
GF290 (+/-)
GF402 /-
GF366 -
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Table 48.
Reagent Target FES 22 FES 30 mEF % stain
FITC-PSA a-Man +
FITC-RCA (3-Ga1 (Gal(34G1cNAc) + - +/-
FITC-PNA (3-Ga1 (Ga1(33GaLNAc) + + -
FITC-MAA a2,3-sialyl-LN + + -
FITC-SNA a2,6-sialyl-LN + n.d. +
FITC-PWA I-antigen + + n.d.
FITC-STA i-antigen + - +
FITC-WFA (3-Ga1NAc + + -
NeuGc-PAA-biotin NeuGc-lectin + + +
anti-GM3(Gc) mAb NeuGca3Ga1(34G1c + + +
FITC-LTA a-Fuc + +
FITC-UEA a-Fuc + - +
mAb Lex Lewis' + n.d. -
mAb sLex sialyl-Lewis" + n.d.
GF 279 Le c Gal(33G1cNAc + - 95-100
GF 283 Le b + - 20-35
GF 284 H Type 2 + - 15-20
GF 285 H Type 2 - + 95-100
GF 286 H Type 2 + - 10-20
GF 287 H Type 1 + - 90-100
GF 288 Globo-H + - 20-35
GF 289 Ley - + 95-100
GF 290 H Type 2 + - 20-35
+, specific binding.
-, no specific binding.
n.d., not determined.
% of stain means approximate percentage of cell stained with a binder.
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Table 492) FES 21 FES 22 FES 29 FES 30 EB2)
Affymetrix ID Gene Bank ID Gene Det.3) Ch.4) Det. Ch. Det. Ch. Det. Ch. Det.
206109 at NM 000148.1 FUT1 P I P I P I P I A
214088_s_at AW080549 FUT3 M NC A NC A NC A NC A
209892_at AF305083.1 FUT4 P I P I P I P I A
211225_at U27330 FUT5 A NC A NC A NC A NC A
211225_at U27329.1 FUT5 A NC A NC A NC A NC A
210399_x_at U27336.1 FUT6 A NC A NC A NC A NC A
211882_x_at U27331.1 FUT6 1 A NC A NC A NC A NC A
211885xat U27332.1 FUT6 2 A NC A NC A NC A NC A
211465_x_at U27335.1 FUT6 minor A NC A NC A NC A NC A
210506_at U11282.1 FUT7 A NC A NC A NC A NC A
203988 s at NM 004480.1 FUT8 P NC P NC P NC P NC A
207696 at NM 006581.1 FUT9 A NC A NC A NC A NC A
Affymetrix ID Gene Bank ID Gene Det. Ch. Det. Ch. Det. Ch. Det. Ch. Det.
229203 at NM 173593 34GaINAc-T3 A NC A NC A NC A NC A
200016 x at NM 002409 M GAT3 P NC P D P D P D P
208058 s at NM 002409.2 MGAT3 A NC A NC A NC A NC A
209764_at AL022312 p4GIcNAcT A NC A MD A MD A NC A
206435at NM001478.2 GALGT A NC A NC A NC A NC A
206720_at NM_002410.2 MGAT5 A NC A NC A NC A NC A
203102_s_at NM_002408.2 MGAT2 P I P NC P I P I P
201126 s at NM 002406.2 MGAT1 P NC P NC P NC P NC P
219797_at NM_012214.1 GNT4a A NC P NC A NC M NC A
220189_s_at NM_014275.1 GNT4b P D P NC P NC P NC P
Affymetrix ID Gene Bank ID Gene Det. Ch. Det. Ch. Det. Ch. Det. Ch. Det.
204856_at AB049585 p3GIcNAc-T3 A NC A NC A NC A NC A
225612_s_at BE672260 p3GIcNAc-T5 P D P D P D P D P
232337_at XM_091928 p3GIcNAc-T7 P NC P NC P NC P NC A
221240 s at NM 030765.1 3GIcNAc-T4 P NC A NC A NC P NC A
204856_at NM_014256.1 p3GnT3 A NC A NC A NC A NC A
205505_at NM_001490.1 p6GIcNAcT P I P NC P NC A NC A
203188_at NM_006876.1 i p3GIcNAcT P D P D P MD P NC P
211020_at L19659.1 I p6GIcNAcT A NC M NC A NC A NC A
214504_at NM_020469.1 A a3GaINAcT A NC A NC A NC A NC A
211812_s_at AB050856.1 globosideT P NC A NC P NC P NC A
221131 at NM_016161.1 a,4GIcNAcT M NC P NC P NC M NC A
Affymetrix ID Gene Bank ID Gene Det. Ch. Det. Ch. Det. Ch. Det. Ch. Det.
221935_s_at AER61 P I P I P I P I A
225689_at AGO61 P NC P NC P NC P NC P
210571_s_at CMAH A NC A NC A NC A NC A
205518_s_at CMAH A D M NC A D A NC P
213355at ST3GAL6 A NC A NC A NC A NC A
211379_x_at p3GALT3 P D P D P NC P D P
218918_at MAN1C1 P NC P NC P NC P NC P
208450_at LGALS2 A NC A NC A NC A NC A
208949 s at LGALS3 P D P D P D P D P
Data reference: Skottman, H., et al. (2005).
2)EB, embryoid bodies used as reference in calculation of fold changes.
3)Det. (detection) codes: P, present; A, absent; M, medium.
4) Ch. (fold change) codes: I, increased; D, decreased; NC, no change.
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Table 50. hESC-associated glycan groups revealed b y statistical analysis.
Identification Glycan class Preferred Factors
* glycans # included
hESC-1 Large high-mannose type and H(6-9)N2 1-1, 6-1
glucosylated N-glycans H(10-11)N2 A3-3
A7-2
hESC-2 Small low-mannose type N- 1 cans H1N2 1-3
hESC-3 Sialylated and neutral biantennary- H5N4F(1-2) 1-1
size complex-type N-glycans S1H5N4F(0-1) A4-1
H5N4F1
hESC-4 Large neutral or monosialylated H6N5F(O-1) 1-2
complex-type N-glycans S1H7N6F1 A7-1
S(1-2)H6N5F1
S 1 H8N7F 1
S1H7N6F3
hESC-5 Neutral and sialylated small hybrid- H4N3 3-1
type or monoantennaN- 1 cans S1H4N3F1 A3-2
hESC-6 Sialylated complex-type N-glycans S 1 H4N5F(1-2) A3-1
with N>H type non-reducing
terminal HexNAc
hESC-7 Complex-fucosylated complex-type S1H6N5F2 A8-1
N- 1 cans S 1 H5N4F 2-3
* Glycan class having shared molecular structure according to the present
invention.
Preferred glycan signals for detection of the glycan group.
~ Described in detail under factor analysis specifications of the present
invention with this
Factor numbering.
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Table 51. Differentiated cell-associated glycan gro s in statistical analysis.
Identification Glycan class Preferred Factors
* glycans # included
Diff-1 soluble HexNAcl-type glycans H(3-9)N1 1-1
5-1
Diff-2 non-fucosylated low-mannose type H(2-4)N2 1-2
N- l cans
Diff-3 fucosylated low- and high-mannose H(4-6)N2F1 3-2, 5-3
type N-glycans A4-3
A5-2
Diff-4 small high-mannose type N-glycans H5N2 6-1
A7-3
Diff-5 sialylated and neutral complex-type H5N5(FO-1) 2-2
N-glycans with N=H type non- H4N4(FO-2) 3-4
reducing terminal HexNAc H5N5F(1-3) 4-2
S1H5N5 5-2
H5N5F1P1 A4-2
S1H5N5F1A1 A5-4
S 1-2 H6N6F1 A8-2
Diff-6 neutral and sialylated hybrid-type H(5-6)N3(FO-1) 2-3
and monoantennary N-glycans H(2-3)N2F1 3-1
H3N3 4-1
H4N3F2 A5-1
H(2-4)N3F1 A7-1
S1H5N3F 0-1
Diff-7 sulphated or phosphorylated N- H3N4F1P1 A3-1
glycans; preferably including S(0-2)H5N4F1P1
sulphate ester S(0-1)H5N4P1
H4N3P1
S1H4N3F1P1
H4N4P1
S1H5N4F3P1
H6N5F1P1
H6N5F3P1
Diff-8 small disialylated glycans, S2H(2-4)N2F1 A4-1
preferably including disialic acid S2H 2-4 N3F1 A7-2
Diff-9 multisialylated biantennary-size S2H5N4 A8-1
com lex-t e N- 1 cans S2H5N5F1
Diff-10 sialylated and neutral complex-type H4N5 2-1
N-glycans with N>H type non- H4N5F(2-3) 3-3
reducing terminal HexNAc H3N4F(0-1) A4-4
S 1 H5N6F2 A5-3
H3N5F1
Diff-11 0-acetylated sialylatedN-glycans S1H7N5FlAl A8-3
S1H6N4F1A1
*,#, See footnotes of the preceding Table.
